JP2022512401A - Harboxy Diene Splicing Regulatory Antibodies-Drug Conjugates and Their Usage - Google Patents

Abstract

Linker-drug compounds and antibody-drug conjugates that bind to human oncology targets are disclosed. This linker-drug compound and antibody-drug conjugate comprises a harboxydien splicing drug moiety. The disclosure further relates to methods and compositions for use in the treatment of neoplastic disorders by administering the antibody-drug conjugates provided herein. [Selection diagram] None

Description

This disclosure discloses US provisional patent application No. 62 / 779,400 filed December 13, 2018; US provisional patent application No. 62 / 779,406 filed December 13, 2018. Book; and claims priority benefit to US Provisional Patent Application No. 62/941,220, filed November 27, 2019. All of the aforementioned applications are incorporated herein by reference in their entirety.

The present disclosure relates to an antibody-drug conjugate (ADC) comprising a harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof, eg, one that binds to a human oncology antigen target. The present disclosure further relates to methods and compositions useful for the treatment or diagnosis of cancers expressing target antigens and / or cancers to which treatment by disrupting RNA splicing is applicable, and methods of making such compositions.

The majority of protein-coding genes in the human genome are composed of multiple exons (coding regions) separated by introns (non-coding regions). Gene expression results in a single precursor messenger RNA (premRNA). Subsequent removal of the intron sequence from the premRNA by a process called splicing results in mature messenger RNA (mRNA). In alternative splicing, the inclusion of various combinations of exons yields mRNAs that encode individual protein isoforms.

RNA splicing is catalyzed by spliceosomes, a dynamic multiprotein-RNA complex composed of five small nuclear RNAs (snRNA U1, U2, U4, U5, and U6) and related proteins. Spliceosomes assemble on premRNAs to build a dynamic cascade of multiple RNA and protein interactions that catalyze intron excision and exon ligation (Matera and Wang (2014) Nat Rev Mol Cell Biol. 15). (2): 108-21). There is increasing evidence to link human disease to the abnormal regulation of RNA splicing that affects many genes (Scottian and Swanson (2016) Nat Rev Genet. 17 (1): 19-32).

Spliceosomes are important targets in cancer biology. Currently, several studies have demonstrated significant changes in the splicing profile of cancer cells, as well as the splicing factors themselves (Agrawal et al. (2018) Curr Opin Genet Dev. 48: 67-74). Alternative splicing can lead to differences in exon inclusion / removal, intron retention, or utilization of potential splice sites (Seiler et al. (2018) Cell Rep. 23 (1): 282-296). Taken together, these events account for functional changes that can contribute to tumorigenesis or treatment resistance (Siegfried and Karni (2018) Curr Opin Genet Dev. 48: 16-21).

Certain natural products can bind to the SF3b spliceosome complex. These small molecules regulate splicing by enhancing intron retention and / or exon skipping (Teng et al. (2017) Nat Commun. 8: 15522). For example, the naturally occurring polyketide harboxydiene (Isaac et al. (1992) J. Org. Chem. 57: 7220-26) isolated from the Streptomyces sp. A7847 and its derivatives. It has been shown to regulate splicing. For example, Imaizumi et al. (2017) J.M. Antibiot. See 70: 675-79. A significant proportion of the resulting transcripts contain immature stop codons that trigger the nonsense-mediated decay mechanism (NMD). In addition, the impaired canonical splicing results in a significant reduction in canonical transcripts, which can have a negative impact on cell function and viability. This makes splicing regulators a promising drug class for the treatment of cancer (Putenveetil et al. (2016) Bioconjugate Chem. 27: 1880-8).

The proto-oncogene human epidermal growth factor receptor 2 (HER2) encodes a transmembrane tyrosine kinase receptor belonging to the human epidermal growth factor receptor (EGFR) family (King et al. (1985) Science 229: 974- 6). Overexpression of HER2 allows constitutive activation of growth factor signaling pathways such as the PI3K-AKT-mTOR pathway and thus acts as a carcinogenic driver in several cancer types, including about 20% of invasive breast cancers (Slamon). et al. (1989) Science 244: 707-12; Gajria and Chandarlapati (2011) Expert Rev Anticancer Ther. 11: 263-75). Given that HER2 amplification mediates the transforming phenotype, and because HER2 expression is largely confined to malignant cells, HER2 is a targeting and / or novel cancer treatment for certain cancers. It is a promising antigen for delivery (Parak et al. (2017) Cancer Treat Rev. 59: 1-21). Further antigens for targeted cancer therapy delivery include, but are not limited to, CD138 (also known as Cindecane-1) and ephrin type A receptor 2 (EPHA2).

CD138 is a cell surface heparan sulfate proteoglycan essential for maintaining cell morphology and interacting with the surrounding microenvironment (Akl et al. (2015) Oncotarget 6 (30): 28693-715; Szatmari et al. (2015)). Dis Markers 2015: 796052). In general, loss of CD138 expression in cancer cells reduces cell adhesion to the extracellular matrix and enhances cell motility and invasion (Teng et al. (2012) Matrix Biol. 31: 3-16). Increased stromal CD138 expression also alters fibronectin production and extracellular matrix tissue (Yang et al. (2011) Am J Pathol. 178: 325-35). In addition, increased expression of CD138 in stromal fibroblasts is associated with angiogenesis and cancer progression (Maeda et al. (2006) Oncogene 25: 1408-12). CD138 expression is increased during B cell development and its presence is characteristic of plasma cells (Ribatti (2017) Immunol Lett. 188: 64-7). CD138 expression is maintained in multiple myeloma, a malignant tumor of plasma cells. CD138 is therefore an attractive antigen for the targeted treatment of some cancers and other hematological malignancies (Sherbenou et al. (2015) Blood Rev. 29 (2): 81-91; Wijdenes et. al. (1996) Br J Hematol. 94 (2): 318-23).

EPHA2 is a transmembrane glycoprotein that is overexpressed in large amounts in several malignant cancer-derived cell lines and advanced cancers (Wykosky and Devinski (2008) Mol Cancer Ref. 6 (12): 1795-1806). For example, EPHA2 is about 61% of GBM patient tumors (Wykosky et al. (2008) Clin Cancer Res. 14: 199-208) and 76% of ovarian cancers (Thaker et al. (2004) Clin Cancer Res. 10 :. It is strongly overexpressed in 5145-50) and 85% of prostate adenocarcinomas (Zeng et al. (2003) Am J Pathol. 163: 2271-6). The EPHA2 protein is highly overexpressed in proportion to patient tumors and cells within tumors and is a cell membrane localized receptor that can be internalized upon ligand binding (Walker-Daniels et al). (2002) Mol Cancer Res. 1: 79-87). In addition, EPHA2 expression is associated with poor prognosis, increased metastasis, and decreased survival. Therefore, EPHA2 is another attractive antigen for targeted delivery of novel anti-cancer therapies because of its expression pattern, localization, and functional importance in the outcome of cancer patients.

In various embodiments, the present disclosure, in part, provides novel harboxydiene splicing regulators with anti-neoplastic biological activity. This harboxydiene splicing regulator may be used alone or as part of an ADC to slow, inhibit, and / or reverse tumor growth in mammals and is useful in the treatment of human cancer patients. obtain. In various embodiments, the present disclosure provides novel antibody-drug conjugates with harboxydiene splicing regulators.

More specifically, the present disclosure relates to antibody-drug conjugate (ADC) compounds capable of binding to and killing neoplastic cells in various embodiments. In various embodiments, the ADC compounds disclosed herein include a linker that binds a harboxydiene splicing regulator to a full-length antibody or antigen-binding fragment. In various embodiments, the ADC compound also has the ability to internalize to the target cell after binding.

In some embodiments, the antibody-drug conjugate is of formula (I): Ab- (L—H) _p [wherein Ab is an antibody or antigen binding fragment that targets a neoplastic cell; H is a harboxydiene splicing regulator; L is a linker that covalently binds Ab to H; and p is an integer of 1-15] antibody-drug conjugate.

又はその薬学的に許容可能な塩［式中：
Ｙは、Ｏ、Ｓ、ＮＲ^６、及びＣＲ^６Ｒ^７から選択され；
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^４は、水素、Ｃ_１～Ｃ_６アルキル基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、及び－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^５は、水素、ヒドロキシル、－ＣＨ_２－ＯＨ、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、－Ｏ－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、及び－ＮＲ^６－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｒ^５、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され、及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (I) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
Y is selected from O, S, NR ⁶ , and CR ⁶ R ⁷ ;
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁴ is hydrogen, C ₁ to C ₆ alkyl group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C. Selected from (= O)-(C ₃ to C ₈ heterocyclyl) groups and -C (= O) -NR ⁶ R ⁷ ;
R ⁵ is hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C (= O) -O- (C ₁ to C ₆ alkyl) group, -C (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , -OC (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , and -NR ⁶ -C (= O) -Selected from NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ and -C (= O) -OR ⁸ ; and R ⁸ is C ₁ Selected from ~ C ₆ alkyl groups, C ₃ ~ C ₈ carbocyclyl groups, and C ₃ ~ C ₈ heterocyclyl groups.
Here, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and —O— (C ₁ ). -C ₆ alkyl) group, -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ ). -C ₃ alkyl) and -NH-C (= O) -O- (may be independently substituted with 0 or 1 group selected from C ₁ to C ₃ alkyl)). It is substituted with 0 to 3 groups and does not exceed the valence of the atom covalently attached to L here].

又はその薬学的に許容可能な塩［式中：
Ｒ^９は、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され；
Ｒ^１０は、Ｈ及びＣ_１～Ｃ_６アルキル基から選択され、
ここでＲ^９及びＲ^１０は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、－ＮＨ_２、－ＮＨ－（Ｃ_１～Ｃ_３アルキル）、及び－Ｎ－（Ｃ_１～Ｃ_３アルキル）_２から独立に選択される０～３個の基で置換され、及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (Ia) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ⁹ is selected from C ₃ to C ₈ heterocyclyl groups;
R ¹⁰ is selected from H and C ₁ to C ₆ alkyl groups.
Here, R ⁹ and R ¹⁰ are independently halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, -NH ₂ , -NH- (C ₁ to C ₃ alkyl), and -N- (C ₁ to C ₃ alkyl) substituted with 0 to 3 groups independently selected from ₂ , and where the valence of the atom covalently attached to L is not exceeded] including.

又はその薬学的に許容可能な塩［式中：
Ｒ^１１は、

（式中、^＊は、Ｒ^１１が化合物の残りの部分に結合する点を表す）から選択され；
Ｒ^１２及びＲ^１３は、各々独立に、Ｈ及びメチルから選択され；及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (Ib) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ¹¹ is

(In the formula, ^* represents the point where R ¹¹ binds to the rest of the compound);
R ¹² and R ¹³ are each independently selected from H and methyl; and here do not exceed the valence of the atom covalently attached to L].

又はその薬学的に許容可能な塩［式中：
Ｘは、ヒドロキシル又はＮＲ^６Ｒ^７であり；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、－Ｃ（＝Ｏ）－Ｏ－Ｒ^８、－（Ｃ_１～Ｃ_６アルキル）－Ｏ－Ｃ（＝Ｏ）－Ｒ^８、及び－（Ｃ_１～Ｃ_６アルキル）－ＮＨ－Ｃ（＝Ｏ）－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され、及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (II) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
X is hydroxyl or NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently hydrogen, -R ⁸ , -C (= O) -R ⁸ , -C (= O) -OR ⁸ ,-(C ₁ to C ₆ alkyl) -O. -C (= O) -R ⁸ and-(C ₁ to C ₆ alkyl) -NH-C (= O) -R ⁸ ; and R ⁸ is a C ₁ to C ₆ alkyl group, C ₃ Selected from the ~ _{C8 carbocyclyl} group and the C3 ~ _C8 heterocyclyl group.
Here, R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, -O- (C ₁ to C ₆ alkyl) groups, -CO ₂ H, and -C (, respectively. = O) -O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl Groups and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O). ) (C1 to C3 alkyl) and -NH _{- C (} = O) -O- ₍ C1 to C3 alkyl) may be independently substituted with 0 or 1 group selected from them). It is substituted with 0 to 3 groups independently selected from, and does not exceed the valence of the atom covalently attached to L here].

又はその薬学的に許容可能な塩［式中：
Ｚは、ＮＲ^９及びＯから選択され；
Ｒ^９は、水素及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^１０及びＲ^１１は、各々独立に、水素、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；
Ｒ^１２は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^９、Ｒ^１０、Ｒ^１１、及びＲ^１２は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、及びＣ_１～Ｃ_３ハロアルキル基から選択される０又は１個の基で置換され；
ｔは、１、２、３、４、５、及び６から選択される整数であり；及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (IIa) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
Z is selected from NR ⁹ and O;
R ⁹ is selected from hydrogen and C ₁ to C ₆ alkyl groups;
R ¹⁰ and R ¹¹ are independently hydrogen, halogen, hydroxyl, C ₁ to C ₆ alkyl group, -O- (C ₁ to C ₆ alkyl) group, -CO ₂ H, -C (= O)-. O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl group. Selected from;
R ¹² is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups.
Here, R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, and C ₁ to C ₃ haloalkyl group, respectively. Substituted with 0 or 1 group to be;
t is an integer selected from 1, 2, 3, 4, 5, and 6; and here does not exceed the valence of the atom covalently attached to L].

又はその薬学的に許容可能な塩［式中：
Ｒ^１３は、

（式中、^＊は、Ｒ^１３が化合物の残りの部分に結合する点を表す）から選択され；
Ｒ^１４及びＲ^１５は、各々独立に、水素及びメチルから選択され；及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (IIb) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ¹³ is

( ^In the formula, ^* represents the point where R13 binds to the rest of the compound);
R ¹⁴ and R ¹⁵ are each independently selected from hydrogen and methyl; and here do not exceed the valence of the atom covalently attached to L].

又はその薬学的に許容可能な塩［式中：
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；及び
Ｒ^９は、Ｈ、

から選択され；
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され、
ここでＬに共有結合的に結び付いている原子の原子価を超えることはなく；及び
式中、^＊は、Ｒ^９が化合物の残りの部分に結合する点を表す］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (III) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ , and -C (= O) -OR ⁸ ;
R ⁸ is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups; and R ⁹ is H,

Selected from;
Here, R ¹ , R ² , R ³ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and -O- (C ₁ to C ₆ alkyl) groups, respectively. , -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ ) ~ C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ ~ C ₈ carbocyclyl group, C ₁ ~ C ₆ alkyl hydroxy group, C ₁ ~ C ₆ alkyl alkoxy group, benzyl group, and C ₃ ~ C ₈ heterocyclyl group. (Each of these is halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ to C ₃ alkyl), And 0 to 3 independently selected from 0 or 1 group selected from -NH-C (= O) -O- (C ₁ to C ₃ alkyl)). Replaced by the group of
Here it does not exceed the valence of the atom covalently attached to L; and in the equation, ^* represents the point where R ⁹ is attached to the rest of the compound].

In some embodiments, the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety is enzymatically cleavable. In some embodiments, the cleavable peptide moiety or linker comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline (“Val-Cit” or “VC”). In some other embodiments, the amino acid unit comprises valine-alanine (“Val-Ala” or “VA”). In some other embodiments, the amino acid unit comprises glutamic acid-valine-citrulline (“Glu-Val-Cit” or “EVC”). In some other embodiments, the amino acid unit comprises alanine-alanine-asparagine (“Ala-Ala-Asn” or “AAN”).

In some embodiments, the linker comprises a cleavable glucuronide moiety. In some embodiments, the cleavable glucuronide moiety is enzymatically cleavable. In some embodiments, the cleaveable glucuronide moiety is cleaveable by glucuronidase. In some embodiments, the cleavable glucuronide moiety is cleavable by β-glucuronidase.

In some embodiments, the linker comprises at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety comprises-(PEG) _m -where m is an integer of 1-10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl moiety. In some embodiments, the alkyl moiety comprises − (CH ₂ ) _n −, where n is an integer of 1-10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, the spacer unit is attached to the antibody or antigen binding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). In some embodiments, the Mal-spacer unit is reactive with cysteine residues on the antibody or antigen binding fragment. In some embodiments, the Mal-spacer unit is coupled to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment.

In some embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Ala. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Glu-Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises maleimide caproyl (MC).

In some embodiments, the Mal-spacer unit binds an antibody or antigen binding fragment to the cleavable portion of the linker. In some embodiments, the cleavable moiety of the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises MC-Val-Cit. In some embodiments, the linker comprises MC-Val-Ala. In some embodiments, the linker comprises MC-Glu-Val-Cit. In some embodiments, the linker comprises MC-Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises maleimide caproyl (MC).

In some embodiments, the cleavable portion of the linker is directly tethered to the harboxydiene splicing regulator, or the spacer unit binds the cleavable portion of the linker to the harboxydiene splicing regulator. In some embodiments, cleavage of the conjugate releases the harboxydiene splicing regulator from the antibody or antigen binding fragment and linker. In some embodiments, the spacer unit that binds the cleavable portion of the linker to the harboxydiene splicing regulator is self-sacrificing.

In some embodiments, the spacer unit that binds the cleavable portion of the linker to the harboxydiene splicing regulator comprises p-aminobenzyloxycarbonyl (pABC). In some embodiments, pABC ligates the cleavable portion of the linker to a harboxydiene splicing regulator. In some embodiments, the cleavable moiety of the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pABC. In some other embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

In some embodiments, the spacer unit that binds the cleavable portion of the linker to the harboxydiene splicing regulator comprises p-aminobenzyl (pAB). In some embodiments, pAB binds the cleavable portion of the linker to a harboxydiene splicing regulator. In some embodiments, the cleavable moiety of the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pAB. In some other embodiments, the linker comprises Val-Ala-pAB. In some other embodiments, the linker comprises Glu-Val-Cit-pAB. In some other embodiments, the linker comprises Ala-Ala-Asn-pAB.

In various embodiments, the linker is a non-cleavable linker. In some embodiments, the harboxydiene splicing regulator of the ADC is released by degradation of the antibody or antigen binding fragment. In some embodiments, the linker remains covalently associated with at least one amino acid of the antibody and drug upon internalization by the target cell and degradation within the target cell.

In some embodiments, the linker is a non-cleavable linker containing at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety comprises-(PEG) _m -where m is an integer of 1-10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl moiety. In some embodiments, the alkyl moiety comprises-(CH _{2) n-or-(CH 2) n-O- (CH 2 ) n , where n} is an integer of 1-10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, the spacer unit of the non-cleavable linker is attached to the antibody or antigen binding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). In some embodiments, the Mal-spacer unit is reactive with cysteine residues on the antibody or antigen binding fragment. In some embodiments, the Mal-spacer unit is coupled to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the linker or Mal-spacer unit comprises maleimide caproyl (MC). In some embodiments, the linker or Mal-spacer unit comprises maleimide caproyl (MC) and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises MC- (PEG) ₂ . In some embodiments, the linker or Mal-spacer unit comprises MC- (PEG) ₂ and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the Mal-spacer unit binds an antibody or antigen binding fragment to a harboxydiene splicing regulator.

In some embodiments, Ab is selected from any of the antibodies or binding domain sequences disclosed herein. In some embodiments, Ab is an antibody or binding domain sequence that targets HER2 and / or neoplastic cells expressing HER2. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing CD138 and / or CD138. In some embodiments, Ab is an antibody or binding domain sequence that targets a neoplastic cell expressing EPHA2 and / or EPHA2. In some embodiments, Ab is an antibody or binding domain sequence that targets a neoplastic cell expressing MSLN and / or MSLN. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing FOLH1 and / or FULLH1. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing CDH6 and / or CDH6. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing CEACAM5 and / or CEACAM5. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing CFC1B and / or CFC1B. In some embodiments, Ab is an antibody or binding domain sequence that targets a neoplastic cell expressing ENPP3 and / or ENPP3. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing FOLR1 and / or FOLR1. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing HAVCR1 and / or HAVCR1. In some embodiments, Ab is an antibody or binding domain sequence that targets KIT and / or neoplastic cells expressing KIT. In some embodiments, Ab is an antibody or binding domain sequence that targets MET and / or neoplastic cells expressing MET. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing MUC16 and / or MUC16. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing SLC39A6 and / or SLC39A6. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing SLC44A4 and / or SLC44A4. In some embodiments, Ab is an antibody or binding domain sequence that targets neoplastic cells expressing STEAP1 and / or STEP1. In some embodiments, Ab is an antibody or binding domain sequence that targets another cancer antigen.

In some embodiments, L is selected from any of the linkers disclosed herein, or any combination of linker components disclosed herein. In some embodiments, L is MC-Val-Cit-pABC, Mal- (PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB. , Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may also include one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15, ADL21, ADL22, or ADL23 linker. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker may also include one or more additional spacer units. In some embodiments, L is an ADL1 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL2 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL5 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL6 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL7 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL12 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL14 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL15 linker and may optionally include one or more additional spacer units. In various embodiments of the ADCs described herein, p is 1-10. In various embodiments, p is 2-8. In various embodiments, p is 4-8. In some embodiments, p is 4. In some embodiments, p is 8.

In some embodiments, pools of ADCs with random conjugation are provided, with an average p in the pool of about 2 to about 8. In some embodiments, pools of ADCs with random conjugation are provided, with an average p in the pool of about 4 to about 8. In some embodiments, a pool of ADCs with random conjugation is provided, with an average p in the pool of about 4. In some embodiments, a pool of ADCs with random conjugation is provided, with an average p in the pool of about 8. A composition (eg, pharmaceutical composition) comprising multiple copies of any of the ADCs described, wherein the average drug loading (mean p) of the ADC in the composition is from about 3.5 to about 5.5. Compositions (eg, about 4), or about 7 to about 9 (eg, about 8), are provided herein.

In some embodiments, the antibody or antigen binding fragment (Ab) of the ADC targets neoplastic cells derived from hematological malignancies or solid tumors. In some embodiments, the antibody or antigen binding fragment targets neoplastic cells derived from hematological malignancies. In some embodiments, the hematological malignancies are selected from B-cell malignancies, leukemias (eg, acute myeloma), lymphomas, and myelomas (eg, multiple myeloma). In some embodiments, the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the antibody or antigen binding fragment targets neoplasmic cells derived from solid tumors. In some embodiments, solid tumors are breast cancer (eg, HER2-positive breast cancer), gastric cancer (eg, gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous). Endometrial cancer), salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colon-rectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In various embodiments, the antibody or antigen binding fragment (Ab) of the ADC is an anti-HER2 antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to HER2 and targets HER2-expressing neoplastic cells (ie, this ADC targets HER2-expressing neoplastic cells). In some embodiments, the antibody or antigen binding fragment of the ADC is an internalizing anti-HER2 antibody or an internalizing antigen binding fragment thereof.

In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3). With (HCDR1, HCDR2, and HCDR3); three light chain complementarity determining regions (LCDR1, LCDR2, and) containing the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3). LCDR3) and included. In some embodiments, the anti-HER2 antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human framework sequence. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-HER2 antibody or antigen binding competes for binding to the same epitope as the antibody comprising the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 20 and / or it. Join.

In various embodiments, the antibody or antigen binding fragment (Ab) of the ADC is an anti-CD138 antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to CD138 and targets neoplastic cells expressing CD138 (ie, this ADC targets neoplastic cells expressing CD138). In some embodiments, the antibody or antigen binding fragment of the ADC is an internalizing anti-CD138 antibody or an internalizing antigen binding fragment thereof.

In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3). With (HCDR1, HCDR2, and HCDR3); three light chain complementarity determining regions (LCDR1, LCDR2, and) containing the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3). LCDR3) and included. In some embodiments, the anti-CD138 antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human framework sequence. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises the mouse Ig kappa light chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises the human IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding competes for binding to the same epitope as the antibody comprising the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 22 and / or it. Join.

In various embodiments, the antibody or antigen binding fragment (Ab) of the ADC is an anti-EPHA2 antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to EPHA2 and targets EPHA2-expressing neoplastic cells (ie, this ADC targets EPHA2-expressing neoplastic cells). In some embodiments, the antibody or antigen binding fragment of the ADC is an internalizing anti-EPHA2 antibody or an internalizing antigen binding fragment thereof.

In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3). With (HCDR1, HCDR2, and HCDR3); three light chain complementarity determining regions (LCDR1, LCDR2, and) containing the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3). LCDR3) and included. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a human framework sequence. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen binding competes for binding to the same epitope as the antibody comprising the heavy chain variable domain of SEQ ID NO: 23 and the light chain variable domain of SEQ ID NO: 24, and / or it. Join.

又はその薬学的に許容可能な塩［式中：
Ｙは、Ｏ、Ｓ、ＮＲ^６、及びＣＲ^６Ｒ^７から選択され；
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^４は、水素、Ｃ_１～Ｃ_６アルキル基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、及び－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^５は、水素、ヒドロキシル、－ＣＨ_２－ＯＨ、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、－Ｏ－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、及び－ＮＲ^６－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｒ^５、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換される］が開示される。 In some embodiments, the compound of formula (I) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
Y is selected from O, S, NR ⁶ , and CR ⁶ R ⁷ ;
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁴ is hydrogen, C ₁ to C ₆ alkyl group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C. Selected from (= O)-(C ₃ to C ₈ heterocyclyl) groups and -C (= O) -NR ⁶ R ⁷ ;
R ⁵ is hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C (= O) -O- (C ₁ to C ₆ alkyl) group, -C (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , -OC (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , and -NR ⁶ -C (= O) -Selected from NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ and -C (= O) -OR ⁸ ; and R ⁸ is C ₁ Selected from ~ C ₆ alkyl groups, C ₃ ~ C ₈ carbocyclyl groups, and C ₃ ~ C ₈ heterocyclyl groups.
Here, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and —O— (C ₁ ). -C ₆ alkyl) group, -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ ). -C ₃ alkyl) and -NH-C (= O) -O- (may be independently substituted with 0 or 1 group selected from C ₁ to C ₃ alkyl)). It is substituted with 0 to 3 groups to be substituted] is disclosed.

又はその薬学的に許容可能な塩［式中：
Ｒ^９は、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され；及び
Ｒ^１０は、Ｈ及びＣ_１～Ｃ_６アルキル基から選択され、
ここでＲ^９及びＲ^１０は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、－ＮＨ_２、－ＮＨ－（Ｃ_１～Ｃ_３アルキル）、及び－Ｎ－（Ｃ_１～Ｃ_３アルキル）_２から独立に選択される０～３個の基で置換される］が提供される。 In some embodiments, the compounds of formula (Ia) are described herein:

Or its pharmaceutically acceptable salt [in the formula:
R ⁹ is selected from the C ₃ to C ₈ heterocyclyl groups; and R ¹⁰ is selected from the H and C ₁ to C ₆ alkyl groups.
Here, R ⁹ and R ¹⁰ are independently halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, -NH ₂ , -NH- (C ₁ to C ₃ alkyl), and -N- (C ₁ to C ₃ alkyl) ₂ substituted with 0 to 3 groups independently selected] is provided.

又はその薬学的に許容可能な塩［式中：
Ｒ^１１は、

（式中、^＊は、Ｒ^１１が化合物の残りの部分に結合する点を表す）から選択され；及び
Ｒ^１２及びＲ^１３は、各々独立に、Ｈ及びメチルから選択される］が提供される。 In some embodiments, the compounds of formula (Ib) are described herein:

Or its pharmaceutically acceptable salt [in the formula:
R ¹¹ is

(In the formula, ^* represents the point where R ¹¹ binds to the rest of the compound); and R ¹² and R ¹³ are independently selected from H and methyl, respectively]. ..

又はその薬学的に許容可能な塩［式中：
Ｘは、ＮＲ^６Ｒ^７であり；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、－Ｃ（＝Ｏ）－Ｏ－Ｒ^８、－（Ｃ_１～Ｃ_６アルキル）－Ｏ－Ｃ（＝Ｏ）－Ｒ^８、及び－（Ｃ_１～Ｃ_６アルキル）－ＮＨ－Ｃ（＝Ｏ）－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換される］が提供される。 In some embodiments, the compound of formula (II) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
X is NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently hydrogen, -R ⁸ , -C (= O) -R ⁸ , -C (= O) -OR ⁸ ,-(C ₁ to C ₆ alkyl) -O. -C (= O) -R ⁸ and-(C ₁ to C ₆ alkyl) -NH-C (= O) -R ⁸ ; and R ⁸ is a C ₁ to C ₆ alkyl group, C ₃ Selected from the ~ _{C8 carbocyclyl} group and the C3 ~ _C8 heterocyclyl group.
Here, R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, -O- (C ₁ to C ₆ alkyl) groups, -CO ₂ H, and -C (, respectively. = O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl groups, C ₁ to C ₆ alkyl hydroxy groups, C ₁ to C ₆ alkyl alkoxy groups, benzyl groups, and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, C ₁ to C ₃ alkyl groups, C ₁ to C ₃ alkoxy groups, C ₁ to C ₃ haloalkyl groups, -NH-C (= O) (C ₁ to C ₃ alkyl), and -NH-C (= O). -Substituted with ₀ to ₃ groups independently selected from 0- (which may be independently substituted with 0 or 1 group selected from C1 to C3 alkyl)] is provided. Will be done.

又はその薬学的に許容可能な塩［式中：
Ｚは、ＮＲ^９及びＯから選択され；
Ｒ^９は、水素及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^１０及びＲ^１１は、各々独立に、水素、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；
Ｒ^１２は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^９、Ｒ^１０、Ｒ^１１、及びＲ^１２は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、及びＣ_１～Ｃ_３ハロアルキル基から選択される０又は１個の基で置換され；及び
ｔは、１、２、３、４、５、及び６から選択される整数である］が提供される。 In some embodiments, the compounds of formula (IIa) are described herein:

Or its pharmaceutically acceptable salt [in the formula:
Z is selected from NR ⁹ and O;
R ⁹ is selected from hydrogen and C ₁ to C ₆ alkyl groups;
R ¹⁰ and R ¹¹ are independently hydrogen, halogen, hydroxyl, C ₁ to C ₆ alkyl group, -O- (C ₁ to C ₆ alkyl) group, -CO ₂ H, -C (= O)-. O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl group. Selected from;
R ¹² is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups.
Here, R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, and C ₁ to C ₃ haloalkyl group, respectively. Substituted with 0 or 1 group to be; and t is an integer selected from 1, 2, 3, 4, 5, and 6] is provided.

又はその薬学的に許容可能な塩［式中：
Ｒ^１３は、

（式中、^＊は、Ｒ^１３が化合物の残りの部分に結合する点を表す）から選択され；及び
Ｒ^１４及びＲ^１５は、各々独立に、水素及びメチルから選択される］が提供される。 In some embodiments, the compound of formula (IIb) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
R ¹³ is

(In the formula, ^* represents the point where R ¹³ binds to the rest of the compound); and R ¹⁴ and R ¹⁵ are independently selected from hydrogen and methyl, respectively]. ..

又はその薬学的に許容可能な塩［式中：
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；及び
Ｒ^９は、Ｈ、

から選択され；
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され；及び
式中、^＊は、Ｒ^９が化合物の残りの部分に結合する点を表す］が提供される。 In some embodiments, the compound of formula (III) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ , and -C (= O) -OR ⁸ ;
R ⁸ is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups; and R ⁹ is H,

Selected from;
Here, R ¹ , R ² , R ³ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and -O- (C ₁ to C ₆ alkyl) groups, respectively. , -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ ) ~ C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ ~ C ₈ carbocyclyl group, C ₁ ~ C ₆ alkyl hydroxy group, C ₁ ~ C ₆ alkyl alkoxy group, benzyl group, and C ₃ ~ C ₈ heterocyclyl group. (Each of these is halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ to C ₃ alkyl), And 0 to 3 independently selected from 0 or 1 group selected from -NH-C (= O) -O- (C ₁ to C ₃ alkyl)). And in the formula, ^* represents the point where ^R9 binds to the rest of the compound] is provided.

及びその薬学的に許容可能な塩から選択される化合物
［式中、Ｌは、抗体に共有結合的に結び付いているリンカーである］が提供される。 In some embodiments, the specification will include.

And a compound selected from pharmaceutically acceptable salts thereof [in the formula, L is a linker covalently attached to the antibody].

Also, in various embodiments, the present specification also provides therapeutic use for the described ADC compounds, harboxidiene compounds, and compositions, for example in the treatment of neoplastic disorders, such as cancer. In certain embodiments, the present disclosure discloses antibodies or antigens of an ADC such as HER2, CD138, EPHA2, MSLN, FULLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1. Provided are methods for treating neoplastic disorders that express antigens targeted by binding fragments, such as cancer.

In certain embodiments, the present disclosure administers a subject having or suspected of having a neoplastic disorder to a therapeutically effective amount and / or therapeutically effective regimen of any one of the described ADCs or compositions. Provides a method of treatment with. In some embodiments, the neoplastic disorder is a hematological malignancies or solid tumors. In some embodiments, the neoplastic disorder is a hematological malignancies. In some embodiments, the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. In some embodiments, the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the neoplastic disorder is a solid tumor. In some embodiments, solid tumors are breast cancer (eg, HER2-positive breast cancer), gastric cancer (eg, gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous). Endometrial cancer), salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colon-rectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic cells adjacent to neoplastic cells that do not express the target antigen but express the target antigen. In some embodiments, the subject has one or more neoplastic cells expressing the target antigen.

In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplasmic cells are HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous endometrial cancer), bone. If you have sarcoma or salivary duct cancer. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-HER2 antibody when administered alone and / or (b) a harboxydiene splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a harboxydiene splicing regulator when administered alone.

In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are in multiple myeloma expressing CD138. In some embodiments, the subject is refractory or refractory to treatment with (a) anti-CD138 antibody when administered alone and / or (b) a harboxydiene splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a harboxydiene splicing regulator when administered alone.

In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are in breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that express EPHA2. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-EPHA2 antibody when administered alone and / or (b) a harboxydiene splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a harboxydiene splicing regulator when administered alone.

In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are in ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) expressing MSLN. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-MSLN antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplasmic cells are in prostate cancer expressing FOLH1. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-FOLH1 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplasmic cells are in renal cancer expressing CDH6. In some embodiments, the subject is refractory or refractory to treatment with (a) anti-CDH6 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplastic cells are in colorectal cancer expressing CEACAM5. In some embodiments, the subject is refractory or refractory to treatment with (a) anti-CEACAM5 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplasmic cells are in pancreatic cancer expressing CFC1B. In some embodiments, the subject is refractory or refractory to treatment with (a) anti-CFC1B antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplasmic cells are in renal cell carcinoma expressing ENPP3. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-ENPP3 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplasmic cells are in ovarian cancer expressing FOLR1. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-FOLR1 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are in renal or esophageal cancer expressing HAVCR1. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-HAVCR1 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplasmic cells are in renal cancer expressing KIT. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-KIT antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are in renal or esophageal cancer expressing MET. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-MET antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplasmic cells are in ovarian, cervical, or breast cancer expressing MUC16. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-MUC16 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplasmic cells are in breast or prostate cancer expressing SLC39A6. In some embodiments, the subject is refractory or refractory to treatment with (a) anti-SLC39A6 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplasmic cells are in prostate cancer expressing SLC44A4. In some embodiments, the subject is refractory or refractory to treatment with (a) an anti-SLC44A4 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more neoplasmic cells are in prostate cancer expressing STEAP1. In some embodiments, the subject is refractory or refractory to treatment with (a) anti-STEAP1 antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, intolerant, or refractory to treatment with a splicing regulator when administered alone.

In certain other embodiments, the present disclosure relates to a therapeutically effective amount and / or a therapeutically effective regimen of any one of the described ADCs or compositions in a subject having or suspected of having a neoplastic disorder. Provide a method for reducing or inhibiting the growth of a tumor.

In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic tumor cells flanking the neoplastic tumor cells that do not express the target antigen but express the target antigen. .. In some embodiments, the tumor comprises one or more neoplasmic cells expressing the target antigen.

In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplasmic cells are HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous endometrial cancer), bone. Derived from sarcoma or salivary duct cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HER2 antibody when administered alone and / or (b) a harboxydiene splicing regulator when administered alone.

In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from multiple myeloma expressing CD138. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-CD138 antibody when administered alone and / or (b) a harboxydiene splicing regulator when administered alone.

In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that express EPHA2. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-EPHA2 antibody when administered alone and / or (b) a harboxydiene splicing regulator when administered alone.

In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) expressing MSLN. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MSLN antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplasmic cells are derived from prostate cancer expressing FOLH1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLH1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplasmic cells are derived from renal cell carcinoma expressing CDH6. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-CDH6 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplasmic cells are derived from colorectal cancer expressing CEACAM5. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-CEACAM5 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is CFC1B. In some embodiments, one or more neoplasmic cells are derived from pancreatic cancer expressing CFC1B. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-CFC1B antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is ENPP3. In some embodiments, one or more neoplasmic cells are derived from renal cell carcinoma expressing ENPP3. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-ENPP3 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplasmic cells are derived from ovarian cancer expressing FOLR1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLR1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplasmic cells are derived from renal or esophageal cancer expressing HAVCR1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HAVCR1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplasmic cells are derived from renal cell carcinoma expressing KIT. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-KIT antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are derived from MET-expressing renal or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MET antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplasmic cells are derived from ovarian cancer, cervical cancer, or breast cancer expressing MUC16. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-MUC16 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplasmic cells are derived from breast or prostate cancer expressing SLC39A6. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-SLC39A6 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplasmic cells are derived from prostate cancer expressing SLC44A4. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-SLC44A4 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplasmic cells are derived from prostate cancer expressing STEAP1. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-STEAP1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

In yet another embodiment, the present disclosure is a biological sample from a subject that has or is suspected of having a neoplastic disorder and whether treatment with any one of the ADCs or compositions described is effective. And a method of determining by contacting the biological sample with an ADC or composition. In some embodiments, the biological sample is a tumor sample. In some embodiments, the tumor sample is a tumor biopsy or blood sample. In some embodiments, the blood sample is selected from blood, blood fractions, or cells obtained from blood or blood fractions. In some embodiments, the subject has one or more neoplastic cells expressing the target antigen. In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplasmic cells are HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous endometrial cancer), bone. Derived from sarcoma or salivary duct cancer. In some embodiments, the target antigen is CD138. In some embodiments, one or more neoplastic cells are derived from multiple myeloma expressing CD138. In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that express EPHA2. In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) expressing MSLN. In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplasmic cells are derived from prostate cancer expressing FOLH1. In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplasmic cells are derived from renal cell carcinoma expressing CDH6. In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplasmic cells are derived from colorectal cancer expressing CEACAM5. In some embodiments, the target antigen is CFC1B. In some embodiments, one or more neoplasmic cells are derived from pancreatic cancer expressing CFC1B. In some embodiments, the target antigen is ENPP3. In some embodiments, one or more neoplasmic cells are derived from renal cell carcinoma expressing ENPP3. In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplasmic cells are derived from ovarian cancer expressing FOLR1. In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are derived from renal or esophageal cancer expressing HAVCR1. In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplasmic cells are derived from renal cell carcinoma expressing KIT. In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are derived from MET-expressing renal or esophageal cancer. In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplasmic cells are derived from ovarian cancer, cervical cancer, or breast cancer expressing MUC16. In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplasmic cells are derived from breast or prostate cancer expressing SLC39A6. In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplasmic cells are derived from prostate cancer expressing SLC44A4. In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplasmic cells are derived from prostate cancer expressing STEAP1.

Further provided herein are pharmaceutical compositions comprising ADCs and pharmaceutically acceptable diluents, carriers, and / or excipients in various embodiments. Also disclosed are methods of making the described ADC compounds and compositions.

1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compound for 72 hours (3 days) or 144 hours (6 days) and survival was read with CellTiter-Glo® 2.0 reagent. FIG. 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. FIG. 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. FIG. 1C shows the viability dose response of HCC 1954 cells after incubation for 144 hours. FIG. 1D shows the viability dose response of NCI-N87 cells after incubation for 144 hours. The data is expressed as mean ± SD. 1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compound for 72 hours (3 days) or 144 hours (6 days) and survival was read with CellTiter-Glo® 2.0 reagent. FIG. 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. FIG. 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. FIG. 1C shows the viability dose response of HCC 1954 cells after incubation for 144 hours. FIG. 1D shows the viability dose response of NCI-N87 cells after incubation for 144 hours. The data is expressed as mean ± SD. 1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compound for 72 hours (3 days) or 144 hours (6 days) and survival was read with CellTiter-Glo® 2.0 reagent. FIG. 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. FIG. 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. FIG. 1C shows the viability dose response of HCC 1954 cells after incubation for 144 hours. FIG. 1D shows the viability dose response of NCI-N87 cells after incubation for 144 hours. The data is expressed as mean ± SD. 1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compound for 72 hours (3 days) or 144 hours (6 days) and survival was read with CellTiter-Glo® 2.0 reagent. FIG. 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. FIG. 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. FIG. 1C shows the viability dose response of HCC 1954 cells after incubation for 144 hours. FIG. 1D shows the viability dose response of NCI-N87 cells after incubation for 144 hours. The data is expressed as mean ± SD. 2A and 2B show the results of the SLC25A19 splicing assay on HER2-amplified breast cancer cells (HCC1954) (FIG. 2A) and gastric cancer cells (NCI-N87) (FIG. 2B). Cells were incubated with the compound for 6 hours and splicing of the SLC25A19 transcript was measured by a real-time qPCR reaction with a specific Taqman primer-probe set. The y-axis represents the percent (%) response to DMSO control (0.1%). The data is expressed as mean ± SD. 2A and 2B show the results of the SLC25A19 splicing assay on HER2-amplified breast cancer cells (HCC1954) (FIG. 2A) and gastric cancer cells (NCI-N87) (FIG. 2B). Cells were incubated with the compound for 6 hours and splicing of the SLC25A19 transcript was measured by a real-time qPCR reaction with a specific Taqman primer-probe set. The y-axis represents the percent (%) response to DMSO control (0.1%). The data is expressed as mean ± SD.

The disclosed compositions and methods may be more easily understood by reference to the detailed description below.

Throughout this document, the description refers to the composition and how to use the composition. Where the disclosure describes or claims features or embodiments relating to a composition, such features or embodiments are equally applicable to the method of use of the composition. Similarly, where the disclosure describes or claims features or embodiments relating to the use of a composition, such features or embodiments are equally applicable to the composition.

When a range of values is represented, it includes embodiments with any particular value within that range. In addition, references to values described in a range include each and every value within that range. All ranges include their endpoints and can be combined. It will be appreciated that when a value is expressed as an approximation by the use of the predecessor "about", that particular value forms another embodiment. References to a particular number include at least that particular value, unless otherwise explicitly stated in the context. The use of "or" shall mean "and / or" unless otherwise indicated in the specific context in which it is used.

For clarity, it is understood that the particular features of the compositions and methods of the present disclosure described herein in the context of separate embodiments may also be provided in combination with a single embodiment. Should be. Conversely, for brevity, the various features of the compositions and methods of the present disclosure described in the context of a single embodiment may also be provided separately or in any partial combination.

All references cited herein are incorporated by reference for all purposes. In the event of a conflict between the reference and the present specification, the present specification shall prevail.

Definitions Throughout the specification and claims, various terms are used with respect to the aspects described. Unless otherwise indicated, such terms should be given their usual meaning in the art. Other specifically defined terms should be construed to be consistent with the definitions provided herein.

As used herein, the singular forms "a", "an", and "the" include the plural unless expressly specified in the context.

The term "about" or "approximately" in the context of numerical values and ranges will be apparent to those of skill in the art from the teachings contained herein of the desired amount of nucleic acid or polypeptide in the reaction mixture. Refers to a value or range that approximates or is close to the value or range described so that the embodiment can operate as intended, such as having. In some embodiments, about means ± 10% of a numerical amount.

The terms "antibody-drug conjugate", "antibody conjugate", "conjugate", "immunoconjugate", and "ADC" are used interchangeably and one or more therapeutic compounds (eg, harboxydienplising). A regulatory agent) linked to one or more antibody or antigen-binding fragment, general formula: Ab- (L-H) _p (formula I) [in the formula, Ab = antibody or antigen-binding fragment, L. = Linker moiety, H = harboxydien splicing regulator (eg, harboxydiene or a derivative thereof), and p = number of drug moieties per antibody or antigen binding fragment]. ADCs containing harboxydiene splicing regulators may also be more specifically referred to herein as "harboxydiene splicing regulator loading antibodies" or "SMLA". In ADCs that include harboxydiene splicing regulators, "p" refers to the number of harboxydiene splicing regulators linked to an antibody or antigen binding fragment. In some embodiments, the linker L may include a cleavable moiety between the antibody or antigen binding fragment and the harboxydiene splicing regulator. In some embodiments, the linker L may comprise a cleavable moiety that can be bound to either or both of an antibody or antigen binding fragment and a harboxydiene splicing regulator by one or more spacer units. In some embodiments, it is a self-sacrificing spacer unit when the spacer unit binds the cleavable portion to a harboxydiene splicing regulator. In another embodiment, the linker L does not contain a cleavable moiety and is a non-cleavable linker. In some embodiments, the linker L may comprise at least one spacer unit that can be directly linked to an antibody or antigen binding fragment and a harboxydiene splicing regulator. Exemplary cleavable and non-cleavable linkers are described and exemplified herein.

The term "antibody", in the broadest sense, recognizes a target such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combination described above via at least one antigen recognition site within the variable region of an immunoglobulin molecule. And is used to refer to an immunoglobulin molecule that specifically binds to it. The heavy chain of an antibody is composed of a heavy chain variable domain ( _VH ) and a heavy chain constant region ( _CH ). The light chain is composed of a light chain variable domain ( _VL ) and a light chain constant domain ( _CL ). For the purposes of the present application, mature heavy and light chain variable domains each have three complementarity determining regions (CDR1, CDR2 and CDR3) within four framework regions (FR1, FR2, FR3, and FR4). From the N-terminal to the C-terminal, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are included side by side. The "antibody" may be naturally occurring or may be artificial, such as a monoclonal antibody produced by conventional hybridoma techniques. The term "antibody" includes full-length monoclonal and polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F (ab') ₂ , Fv, and single-chain antibodies. Antibodies can be one of five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (eg, isotypes IgG1, IgG2, IgG3, IgG4). The term further refers to human antibodies, chimeric antibodies, humanized antibodies, and antigen recognition sites as long as they exhibit the desired biological activity (eg, internalize into target antigen-expressing cells that bind to the target antigen). Includes any modified immunoglobulin molecule contained.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that occupy that population may be present in small amounts. It is identical except for certain naturally occurring mutations. Monoclonal antibodies are highly specific and target a single antigenic epitope. In contrast, conventional (polyclonal) antibody formulations typically include a large number of antibodies that target (or are specific to) different epitopes. The modifier "monoclonal" indicates the nature that the antibody is obtained from a substantially homogeneous population of antibodies and is interpreted that the antibody must be produced by any particular method. must not. For example, the monoclonal antibodies that will be used in the present disclosure are described in Kohler et al. (1975) It may be made by the hybridoma method first described by Nature 256: 495, or by the recombinant DNA method (see, eg, US Pat. No. 4,816,567). matter). Monoclonal antibodies are also described, for example, in Clackson et al. (1991) Nature 352: 624-8, and Marks et al. (1991) J Mol Biol. It may be isolated from the phage antibody library using the technique described in 222: 581-97.

The monoclonal antibodies described herein are specifically heavy chains and / or portions of light chains that are identical to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass. Or a "chimeric" antibody, which is homologous, while the rest of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies as long as they specifically bind to the target antigen and / or exhibit the desired biological activity.

The term "human antibody" as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human.

As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. In some examples, both the heavy and light chain variable regions correspond to the variable regions of the antibody from one species with the desired specificity, affinity, and activity, while the constant regions are separate. It is homologous to an antibody derived from a species of (eg, human), thereby minimizing the immune response in the latter species.

As used herein, the term "humanized antibody" refers to the form of a non-human (eg, mouse) antibody as well as an antibody comprising sequences from human antibodies. Such antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In general, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, here all or substantially all of the hypervariable loops of non-human immunoglobulins. Corresponding to, all or substantially all of the framework (FR) regions are of human immunoglobulin sequences. The humanized antibody will optionally also include an immunoglobulin constant region (Fc), typically at least a portion of the Fc of human immunoglobulin. Humanized antibodies are made by substituting residues in any of the Fv framework regions and / or replaced non-human residues for antibody specificity, affinity, and / or activity refinement and optimization. It may be further modified.

The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to an antigen (eg, HER2, CD138, EPHA2, MSLN, FULLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, Refers to one or more fragments of an antibody or protein that retains the ability to specifically bind to HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1). The antigen-binding fragment may also retain the ability to internalize to antigen-expressing cells. In some embodiments, the antigen-binding fragment also retains immune effector activity. It has been shown that fragments of full-length antibodies can perform the antigen-binding function of full-length antibodies. Examples of binding fragments within the scope of the term "antigen-binding fragment" or "antigen-binding portion" of an antibody are (i) monovalent fragments consisting of _VL , _VH , _CL , and _CH1 domains. Fab fragment; (ii) F (ab') ₂ fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of _VH and _CH1 domains; iv) Fv fragment consisting of _VL and _VH domains of a single arm of antibody; (v) dAb fragment containing a single variable domain, eg _VH domain (eg, Ward et al. (1989) Nature 341: 544. -6; and see WO 1990/005144); and (vi) isolated complementarity determination regions (CDRs). In addition, the two domains of the Fv fragment, _VL and _VH , are encoded by separate genes, but using recombinant methods, they can be joined together by a synthetic linker and the _VL and _VH regions are paired. It becomes possible to prepare it as a single protein chain (known as a single chain Fv (scFv)) forming a monovalent molecule. For example, Bird et al. (1988) Science 242: 423-6; and Huston et al. (1988) Proc Natl Acad Sci. See USA 85: 5879-83. Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" or "antigen-binding portion" of the antibody and, in the art, can be intracellularized upon binding. Known as an exemplary type of binding fragment (eg, Zhu et al. (2010) 9: 2131-41; He et al. (2010) J Nucl Med. 51: 427-32; and Fitting et al. ( 2015) MAbs 7: 390-402). In certain embodiments, the scFv molecule may be integrated into the fusion protein. Other forms of single chain antibodies, such as diabody, are also included. Diabodies are bivalent bispecific antibodies in which the _VH and _VL domains are expressed on a single polypeptide chain, but are too short to allow pairing between two domains on the same chain. Linkers are used, thus forcing those domains to pair with complementary domains of another strand to create two antigen binding sites (eg, Holliger et al. (1993) Proc Natl Acad Sci. USA 90: 6444). -8; and Poljak et al. (1994) Structure 2: 1121-3). Antigen-binding fragments are obtained using conventional techniques known to those of skill in the art, and such binding fragments are screened for usefulness (eg, binding affinity, internalization) in the same manner as intact antibodies. The antigen-binding fragment may be prepared by cleaving the intact protein, for example by protease or chemical cleavage.

"Internalization", as used herein in connection with an antibody or antigen binding fragment, is an internal compartment, preferably a degradable compartment of a cell, through the lipid bilayer membrane of the cell upon binding to the cell. Refers to an antibody or antigen-binding fragment capable of being incorporated into (ie, "internalized"). For example, an internalizing anti-HER2 antibody has the ability to be taken up into cells after binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen binding fragment used in the ADC disclosed herein targets a cell surface antigen (eg, HER2) and is an internalizing antibody or internalizing antigen binding. It is a fragment (ie, this ADC translocates through the cell membrane after antigen binding). In some embodiments, the internalizing antibody or antigen binding fragment binds to a receptor on the cell surface. An internalizing antibody or an internalizing antigen-binding fragment that targets a receptor on the cell membrane can induce receptor-mediated endocytosis. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is taken up into cells by receptor-mediated endocytosis.

"Non-internalizing" as used herein in connection with an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that remains on the cell surface upon binding to the cell. In some embodiments, the antibody or antigen binding fragment used in the ADC disclosed herein targets a cell surface antigen and is a non-internalizing antibody or non-internalizing antigen binding fragment. (That is, this ADC stays on the cell surface after antigen binding and does not migrate through the cell membrane). In some embodiments, the non-internalizing antibody or antigen-binding fragment binds to a non-internalizing receptor or other cell surface antigen. Exemplary non-internalizing cell surface antigens include, but are not limited to, CA125 and CEA, and antibodies that bind to non-internationalizing antigen targets are also known in the art (eg, for example. Bast et al. (1981) J Clin Invest. 68 (5): 1331-7; Scholler and Urban (2007) Biomark Med. 1 (4): 513-23; and Boudousq et al. (2013) PLoS One 8 (2013) 7): See e69613).

The terms "human epidermal growth factor receptor 2", "HER2", or "HER2 / NEU", as used herein, refer to any natural form of human HER2. The term includes full-length HER2 (eg, UniProt reference sequence: P04626; SEQ ID NO: 31), as well as any form of human HER2 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human HER2, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human HER2. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). HER2 can be isolated from humans or may be produced recombinantly or by synthetic methods.

The term "anti-HER2 antibody" or "antibody that binds to HER2" refers to any form of antibody or fragment thereof that binds, for example, specifically binds to HER2, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to HER2. U.S. Pat. No. 5,821,337 provides exemplary HER2 binding sequences, including exemplary anti-HER2 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-HER2 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody. Trastuzumab (US Pat. No. 5,821,337; Molina et al. (2001) Cancer Res. 61 (12): 4744-9) is an exemplary anti-human HER2 antibody.

The terms "Cindecane-1", "SDC1", or "CD138", as used herein, refer to any natural form of human CD138. The term includes full-length CD138 (eg, UniProt reference sequence: P18827; SEQ ID NO: 32), as well as any form of human CD138 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human CD138, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CD138. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). CD138 can be isolated from humans or may be produced recombinantly or by synthetic methods.

The term "anti-CD138 antibody" or "antibody that binds to CD138" refers to any form of antibody or fragment thereof that binds to, for example, specifically binds to CD138, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to CD138. In some embodiments, the anti-CD138 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody. B-B4 (Tassone et al. (2004) Blood 104: 3688-96) is an exemplary anti-human CD138 antibody.

The term "ephrin type A receptor 2" or "EPHA2" as used herein refers to any natural form of human EPHA2. The term includes full-length EPHA2 (eg, UniProt reference sequence: P29317; SEQ ID NO: 33), as well as any form of human EPHA2 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human EPHA2, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human EPHA2. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). EPHA2 can be isolated from humans or may be produced recombinantly or by synthetic methods.

The term "anti-EPHA2 antibody" or "antibody that binds to EPHA2" refers to any form of antibody or fragment thereof that binds to EPHA2, eg, specifically, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to EPHA2. WO 2007/030642 provides exemplary EPHA2 binding sequences, including exemplary anti-EPHA2 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-EPHA2 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody. 1C1 (Pamphlet 2007/030642; Jackson et al. (2008) Cancer Res. 68 (22): 9637-74) is an exemplary anti-human EPHA2 antibody.

The term "mesotelin" or "MSLN" as used herein refers to any natural form of human MSLN. The term includes full-length MSLNs (eg, UniProt reference sequence: Q13421; SEQ ID NO: 94), as well as any form of human MSLN that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human MSLN, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MSLN. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). MSLN can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-MSLN antibody" or "antibody that binds to MSLN" refers to any form of antibody or fragment thereof that binds to MSLN, eg, specifically binds, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, to MSLN. WO 2011/074621 provides exemplary MSLN binding sequences, including exemplary anti-MSLN antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-MSLN antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody. 11-25, IC14-30, IC7-4, IC17-35 and 2-9 are exemplary anti-human MSLN antibodies.

The term "glutamic acid carboxypeptidase 2" or "FOLH1" as used herein refers to any natural form of human FOLH1. The term includes full-length SOLH1 (eg, UniProt reference sequence: Q04609; SEQ ID NO: 95), as well as any form of human SOLH1 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human FOLH1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human FOLH1. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). FOLH1 can be isolated from humans, or it may be produced recombinantly or by synthetic methods.

The term "anti-FOLH1 antibody" or "antibody that binds to FOLH1" refers to any form of antibody or fragment thereof that binds to, for example, specifically binds to FOLH1, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to FULLH1. WO 2019/012260 and WO 2017/212250 provide exemplary FOLH1 binding sequences, including exemplary anti-FOLH1 antibody sequences, which are incorporated herein by reference. .. In some embodiments, the anti-FOLH1 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody. J591 (deimmunization) is an exemplary anti-human FOLH1 antibody.

The term "cadherin-6" or "CDH6" as used herein refers to any natural form of human CDH6. The term includes full-length CDH6 (eg, UniProt reference sequence: P55285; SEQ ID NO: 96), as well as any form of human CDH6 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human CDH6, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CDH6. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). CDH6 can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-CDH6 antibody" or "antibody that binds to CDH6" refers to any form of antibody or fragment thereof that binds to, for example, specifically binds to CDH6, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to CDH6. WO 2018/185618 provides exemplary CDH6 binding sequences, including exemplary anti-CDH6 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-CDH6 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "carcinoembryonic antigen-related cell adhesion molecule 5" or "CEACAM5", as used herein, refers to any natural form of human CEACAM5. The term includes full-length CEACAM 5 (eg, UniProt reference sequence: P06731; SEQ ID NO: 97), as well as any form of human CEACAM 5 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human CEACAM5, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CEACAM5. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). CEACAM5 can be isolated from humans, or may be made recombinantly or by synthetic methods.

The term "anti-CEACAM5 antibody" or "antibody that binds to CEACAM5" refers to any form of antibody or fragment thereof that binds to CEACAM5, eg, specifically binds, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to CEACAM5. U.S. Patent Application Publication No. 2015/0125386 provides exemplary CEACAM5 binding sequences, including exemplary anti-CEACAM5 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-CEACAM5 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody. hMN14 is an exemplary anti-human CEACAM5 antibody.

The term "cryptic family protein 1B" or "CFC1B" as used herein refers to any natural form of human CFC1B. The term includes full-length CFC1B (eg, UniProt reference sequence: P0CG36; SEQ ID NO: 98), as well as any form of human CFC1B that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human CFC1B, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CFC1B. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). CFC1B can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-CFC1B antibody" or "antibody that binds to CFC1B" refers to any form of antibody or fragment thereof that binds to, for example, specifically binds to CFC1B, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to CFC1B. WO 2002/08A170 provides exemplary CFC1B binding sequences, including exemplary anti-CFC1B antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-CFC1B antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "ectonucleotide pyrophosphatase / phosphodiesterase family member 3" or "ENPP3", as used herein, refers to any natural form of human ENPP3. The term includes full-length ENPP3 (eg, UniProt reference sequence: O14638; SEQ ID NO: 99), as well as any form of human ENPP3 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human ENPP3, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human ENPP3. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). ENPP3 can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-ENPP3 antibody" or "antibody that binds to ENPP3" refers to any form of antibody or fragment thereof that binds to ENPP3, eg, specifically, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to ENPP3. Donate et al. ((2016) Clin Cancer Res. 22 (8): 1989-99) provides exemplary ENPP3 binding sequences, including exemplary anti-ENPP3 antibody sequences, which are incorporated herein by reference. .. In some embodiments, the anti-ENPP3 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "folic acid receptor α" or "FOLR1" as used herein refers to any natural form of human FOLR1. The term includes full-length FOLR1 (eg, UniProt reference sequence: P15328; SEQ ID NO: 100), as well as any form of human FOLR1 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human FOLR1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human FOLR1. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). FOLR1 can be isolated from humans, or may be produced recombinantly or by synthetic methods.

The term "anti-FOLR1 antibody" or "antibody that binds to FOLR1" refers to any form of antibody or fragment thereof that binds to FOLR1, eg, specifically, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to FOLR1. International Publication No. 2005/08431 pamphlet and Coney et al. ((1991) Cancer Res. 51 (22): 6125-32), including an exemplary anti-FOLR1 antibody sequence, provides an exemplary FOLR1 binding sequence, which is incorporated herein by reference. In some embodiments, the anti-FOLR1 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody. Farretuzumab and MOv19 are exemplary anti-human FOLR1 antibodies.

The term "hepatitis A virus cell receptor 1" or "HAVCR1" as used herein refers to any natural form of human HAVCR1. The term includes full-length HAVCR1 (eg, UniProt reference sequence: Q96D42; SEQ ID NO: 101), as well as any form of human HAVCR1 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human HAVCR1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human HAVCR1. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). HAVCR1 can be isolated from humans, or may be produced recombinantly or by synthetic methods.

The term "anti-HAVCR1 antibody" or "antibody that binds to HAVCR1" refers to any form of antibody or fragment thereof that binds to HAVCR1, eg, specifically binds, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, to HAVCR1. Thomas et al. ((2016) Mol Cancer Ther. 15 (12): 2946-54), including an exemplary anti-HAVCR1 antibody sequence, provides an exemplary HAVCR1 binding sequence, which is incorporated herein by reference. .. In some embodiments, the anti-HAVCR1 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "mast / stem cell growth factor receptor KIT" or "KIT" as used herein refers to any natural form of human KIT. The term includes a full-length KIT (eg, UniProt reference sequence: P10721; SEQ ID NO: 102), as well as any form of human KIT that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human KIT, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human KIT. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). The KIT can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-KIT antibody" or "antibody that binds to KIT" refers to any form of antibody or fragment thereof that binds to KIT, eg, specifically, and is a monoclonal antibody (including full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to the KIT. Shi et al. ((2016) Proc Natl Acad Sci USA 113 (33): E4784-93) and Abrams et al. ((2018) Clin Cancer Res. 24 (17): 4297-308) provides exemplary KIT binding sequences, including exemplary anti-KIT antibody sequences, which are incorporated herein by reference. .. In some embodiments, the anti-KIT antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "hepatocyte growth factor receptor" or "MET", as used herein, refers to any natural form of human MET. The term includes full-length METs (eg, UniProt reference sequence: P08581; SEQ ID NO: 103), as well as any form of human MET that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human MET, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MET. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). MET can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-MET antibody" or "antibody that binds to MET" refers to any form of antibody or fragment thereof that binds to, for example, specifically binds to MET, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to MET. Yang et al. ((2019) Acta Pharmacol Sin.) Provides exemplary MET binding sequences, including exemplary anti-MET antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-MET antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "mucin-16" or "MUC16" as used herein refers to any natural form of human MUC16. The term includes full-length MUC16 (eg, UniProt reference sequence: Q8WXI7; SEQ ID NO: 104), as well as any form of human MUC16 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human MUC16, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MUC16. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). MUC16 can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-MUC16 antibody" or "antibody that binds to MUC16" refers to any form of antibody or fragment thereof that binds to MUC16, eg, specifically binds, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to MUC16. Liu et al. ((2016) Ann Oncol. 27 (11): 2124-30) provides exemplary MUC16 binding sequences, including exemplary anti-MUC16 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-MUC16 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "zinc transporter ZIP6" or "SLC39A6" as used herein refers to any natural form of human SLC39A6. The term includes full-length SLC39A6 (eg, UniProt reference sequence: Q13433; SEQ ID NO: 105), as well as any form of human SLC39A6 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human SLC39A6, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human SLC39A6. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). SLC39A6 can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-SLC39A6 antibody" or "antibody that binds to SLC39A6" refers to any form of antibody or fragment thereof that binds to, for example, specifically binds to SLC39A6, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to SLC39A6. Susman et al. ((2014) Mol Cancer Ther. 13 (12): 2991-3000) provides an exemplary SLC39A6 binding sequence, including an exemplary anti-SLC39A6 antibody sequence, which is incorporated herein by reference. .. In some embodiments, the anti-SLC39A6 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "choline transporter-like protein 4" or "SLC44A4" as used herein refers to any natural form of human SLC44A4. The term includes full-length SLC44A4 (eg, UniProt reference sequence: Q53GD3; SEQ ID NO: 106), as well as any form of human SLC44A4 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human SLC44A4, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human SLC44A4. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). SLC44A4 can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-SLC44A4 antibody" or "antibody that binds to SLC44A4" refers to any form of antibody or fragment thereof that binds to, for example, specifically binds to SLC44A4, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to SLC44A4. Mattie et al. ((2016) Mol Cancer Ther. 15 (11): 2679-87) provides exemplary SLC44A4 binding sequences, including exemplary anti-SLC44A4 antibody sequences, which are incorporated herein by reference. .. In some embodiments, the anti-SLC44A4 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

The term "metalloreductase STEAP1" or "STEAP1" as used herein refers to any natural form of human STEP1. The term includes full-length STEAP1 (eg, UniProt reference sequence: Q9UHE8; SEQ ID NO: 107), as well as any form of human STEP1 that can result from cell processing. The term also includes, but is not limited to, functional variants or fragments of human STEP1 including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human STEP1. Includes (ie, mutants and fragments are included unless the context dictates that the term is used to refer only to wild-type proteins). STEAP1 can be isolated from humans or may be made recombinantly or by synthetic methods.

The term "anti-STEAP1 antibody" or "antibody that binds to STEAP1" refers to any form of antibody or fragment thereof that binds to STEAP1, eg, specifically binds, a monoclonal antibody (including a full-length monoclonal antibody), polyclonal. Includes antibodies and biologically functional antibody fragments to the extent that they bind, eg, specifically, bind to STEAP1. WO 2008/052187 provides exemplary STEAP1 binding sequences, including exemplary anti-STEAP1 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-STEAP1 antibody used in the ADC disclosed herein is an internalizing antibody or fragment of an internalizing antibody.

As used herein, the terms "specific", "specificly binds", and "binds specificly" refer to proteins and other biological substances. Refers to the binding reaction between an antibody or antigen-binding fragment (eg, anti-HER2 antibody) and a target antigen (eg, HER2) in a heterologous population. Antibodies can be tested for binding specificity by comparing binding to the appropriate antigen to binding to an irrelevant antigen or antigen mixture under a given set of conditions. If an antibody binds to a suitable antigen with an affinity that is at least 2, 5, 7-fold, preferably 10-fold or more higher than that of an unrelated antigen or antigen mixture, it is considered specific. A "specific antibody" or "target-specific antibody" is one that binds only to the target antigen (eg, HER2) but does not (or exhibits minimal binding) to other antigens. In certain embodiments, the antibody or antigen binding fragment that specifically binds to the target antigen (eg, HER2) is less than 1 × 10 ^-6 M, less than 1 × 10 ^-7 M, less than 1 × 10 ^-8 M, It has a KD of less than 1 × 10 ^-9 M, less than 1 × 10 ^-10 M, less than 1 × 10 ^-11 M, less than 1 × 10 ^-12 _M , or less than 1 × 10 ^-13 M. In certain embodiments, the _KD is 1 pM to 500 pM. In some embodiments, _KD is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

The term "epitope" refers to a portion of an antigen that is recognized by an antibody and has the ability to specifically bind. When the antigen is a polypeptide, the epitope may be formed of continuous amino acids, or the polypeptide may be formed of adjacent discontinuous amino acids by being folded into tertiary structure. The epitopes bound by the antibody are X-ray crystallization for epitope identification by direct visualization of the antigen-antibody complex, as well as monitoring of binding of the antibody to a fragment or mutant variant of the antigen, or of the antibody and antigen. It may be identified using any epitope mapping technique known in the art, including monitoring the exposure of the solvent to various parts. Illustrative strategies used for mapping antibody epitopes include, but are not limited to, array-based oligopeptide scanning, protein-limited degradation, site-specific mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, And mass spectrometry (see, eg, Gershoni et al. (2007) 21: 145-56; and Hager-Bran and Tomer (2005) Expert Rev Protocols 2: 745-56).

Competitive binding and epitope binning can also be used to determine antibodies that share the same or overlapping epitopes. Competitive binding is a cross-blocking assay, such as the assay described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, ^Halloween and Lane (1st ^edition 1988, 2nd edition 2014). .. In some embodiments, a target antigen such as HER2 by a reference antibody or binding protein (eg, a binding protein containing a CDR and / or variable domain selected from those identified in Tables 2-4) in a cross-blocking assay. Binding to at least about 50% (eg, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or in between, depending on the test antibody or binding protein. Competitive binding is identified when it decreases by an arbitrary percentage) and / or vice versa. In some embodiments, competitive binding can result from a shared or similar (eg, partially overlapping) epitope, or a steric hindrance to an antibody or binding protein to a nearby epitope. It can be caused by the disorder (see, for example, Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to fractionate a group of binding proteins that share similar epitopes. For example, binding proteins that compete for binding can be "binned" as a group of binding proteins that have overlapping or neighboring epitopes, while non-competing ones have another binding that does not have overlapping or neighboring epitopes. It is divided into protein groups.

The term " _kon " or "ka" refers to the _on -rate constant for association of an antibody with an antigen to form an antibody / antigen complex. This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assay.

The term "k _off " or "k _d " refers to the off rate constant for antibody dissociation from an antibody / antigen complex. This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assay.

The term " _KD " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. _KD is calculated by _ka / _cd . This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assay.

The terms "p" or "harboxydiene splicing regulator load" or "harboxydiene splicing regulator: antibody ratio" or "harboxydiene splicing regulator vs. antibody ratio" or "HAR" are for each antibody or antigen binding. The number of harboxydiene splicing regulators per fragment, i.e., the harboxydiene splicing regulator loading, or the -L-H portion per antibody or antigen binding fragment (Ab) in the ADC disclosed herein. Refers to the number of. In ADCs that include harboxydiene splicing regulators, "p" refers to the number of harboxydiene splicing regulators linked to an antibody or antigen binding fragment. For example, p = 2 when two harboxidien splicing regulators (eg, two compounds each having the structure of H3) are linked to an antibody or antigen binding fragment. In a composition comprising multiple copies of ADC as described herein, "mean p" refers to the average number of —L—H moieties per antibody or antigen binding fragment and is “mean harboxy”. It is also called "diene splicing regulator load".

A "linker" or "linker moiety" as used herein is the ability to covalently bind a drug moiety, such as a compound, usually a harboxydiene splicing regulator drug moiety, to another moiety, such as an antibody or antigen binding fragment. Used to refer to any chemical moiety that has. The linker may be susceptible to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage, provided that the compound or antibody remains active, or substantially It may be resistant.

The term "drug" is used herein to refer to an extract made from a chemical compound, a mixture of chemical compounds, a biological macromolecule, or a biological material. The term "therapeutic agent" or "drug" refers to a drug that has the ability to regulate biological processes and / or has biological activity. The harboxydiene splicing regulators described herein are exemplary therapeutic agents.

The term "chemotherapeutic agent" or "anti-cancer agent" is used herein to refer to any agent effective in treating cancer, regardless of its mechanism of action. Inhibition of metastasis or angiogenesis is a frequent characteristic of chemotherapeutic agents. Chemotherapeutic agents include antibodies, biomolecules, and small molecules, including the harboxydiene splicing regulators described herein. The chemotherapeutic agent can be a cytotoxic agent or a cell proliferation inhibitor. The term "cell proliferation inhibitor" refers to an agent that inhibits or suppresses cell growth and / or cell proliferation. The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with cell expression activity and / or function.

As used herein, the terms "harboxydiene spliceosome regulator", "harboxydiene spliceosome regulator", or "harboxydiene spliceosome regulator" interact with components of the spliceosome. Thereby, it refers to a compound having anticancer activity and structurally related to harboxidiene. In some embodiments, the harboxydiene splicing regulator alters the rate or morphology of splicing in target cells. Harboxydiene splicing regulators that act as inhibitory agents have, for example, the ability to reduce uncontrolled cell proliferation. In some embodiments, the harboxydiene splicing regulator may act by binding to the SF3b spliceosome complex. Such regulators may be naturally occurring, or may be synthetic derivatives or analogs of harboxidiene. As used herein, the terms "derivatives" and "analogs" as used to refer to harboxydien splicing regulators and the like are essentially the same, similar, or enhanced biological functions as harboxydiene. Any such compound that retains its activity but whose chemical or biological structure has changed means. In some embodiments, the harboxydiene splicing regulator is a harboxydiene derivative.

As used herein, the "harboxydiene splicing regulatory drug moiety" is in an ADC or component of the composition that provides the structure of the harboxydiene splicing regulator compound, eg, in the ADC of formula (I). , Or refers to the Harboxidiene Splicing Modulator (H) component in a composition comprising —L—H.

As used herein, "spliceosome" refers to a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

The term "homology" refers to a molecule that exhibits homology with another molecule, for example by having a sequence of chemical residues that are the same or similar at the corresponding positions.

The term "inhibiting" or "inhibiting", as used herein, means reducing by a measurable amount and may include, but is not required, complete prevention or inhibition.

The terms "target negative", "target antigen negative", or "antigen negative" refer to the absence of target antigen expression by cells or tissues. The terms "target positive", "target antigen positive", or "antigen positive" refer to the presence of target antigen expression. For example, a cell or cell line that does not express the target antigen may be described as target negative, while a cell or cell line that expresses the target antigen may be described as target positive.

The term "bystander killing" or "bystander effect" refers to the killing of target-negative cells in the presence of target-positive cells, where no killing of target-negative cells is observed in the absence of target-positive cells. Cell-cell contact between target-positive cells and target-negative cells, or at least close proximity, allows bystander killing. This type of killing is distinguishable from "off-target killing," which refers to the indiscriminate killing of target-negative cells. "Off-target killing" can also be observed in the absence of target-positive cells.

The terms "neoplastic disorder" and "cancer" are used herein to cause cancer, such as uncontrolled growth, immortality, metastatic potential, rapid growth and growth rate, and / or certain morphological features. It is used synonymously to refer to the presence of cells with characteristics typical of cells. In many cases, cancer cells can be in the form of tumors or masses, but such cells may be present alone in the subject or in the bloodstream as independent cells, such as leukemia cells or lymphoma cells. It may be circulated. The terms "neoplastic disorders" and "cancers" include all types of cancers and cancer metastases, including hematological malignancies, solid tumors, sarcomas, carcinomas and other solid and non-solid tumor cancers. Hematological malignancies can include B-cell malignancies, blood cancers (leukemias), plasma cell cancers (myeloma, eg multiple myeloma), or lymph node cancers (lymphomas). Exemplary B-cell malignant tumors include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemia includes acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CML), chronic myelomonocytic leukemia (CMML), Acute monocytic leukemia (AMOL) and the like may be included. Lymphoma may include Hodgkin lymphoma and non-Hodgkin lymphoma. Other hematological malignancies may include myelodysplastic syndrome (MDS). Solid tumors include adenocarcinomas such as breast cancer, pancreatic cancer, prostate cancer, colon or rectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, glioma, melanoma, etc. Cancer may be included.

The terms "tumor" and "neoplasm" refer to any tissue mass resulting from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.

The terms "tumor cells" and "neoplasmic cells" are used synonymously to refer to individual or whole cell populations derived from a tumor or neoplasm, including both non-tumorogenic cells and cancer stem cells. As used herein, the term "tumor cell" is modified by the term "non-tumorogenic" when simply referring to a tumor cell that lacks the ability to regenerate and differentiate, and such tumor cell is referred to as a cancer stem cell. It will be distinguished.

The terms "subject" and "patient" are used interchangeably herein to refer to any animal, including, but not limited to, any mammal, including humans, non-human primates, rodents, and the like. Tooth. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

The term "co-administration" or "in combination" with one or more therapeutic agents includes simultaneous administration and continuous administration in any order.

A "pharmaceutical composition" is a preparation in a form that allows administration and subsequently provides the intended biological activity of one or more active ingredients and / or achieves a therapeutic effect. It refers to a preparation that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is to be administered. The pharmaceutical composition can be sterile.

"Pharmaceutical excipients" include materials such as adjuvants, carriers, pH regulators and buffers, tonicity agents, wetting agents, preservatives and the like.

"Pharmaceutically acceptable" means that its use in animals, more specifically in humans, has been or is expected to be approved by federal or state government regulators, or is the United States Pharmacopeia or other generally accepted pharmacy. It means that it is published in the direction.

A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound and does not give an undesired toxicological effect. Examples of such salts are (a) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitrate and the like; and organic acids such as acetic acid, oxalic acid, tartrate acid, succinic acid. Acids, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonate Salts formed from acids such as acids, polygalacturonic acids; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine. For example, Haynes et al. "Commentary: Occurrence of Pharmaceutically Accessable Anions and Cation in the Cambridge Structural Database," J Physical Science. 94, no. 10 (2005), and Berge et al. "Pharmaceutical Salts," J Pharmaceutical Sciences, vol. 66, no. 1 (1977), which are incorporated herein by reference.

As used herein, the term "effective amount" is sufficient to serve the purposes specifically described, eg, after administration, reduction of tumor growth rate or tumor volume, reduction of cancer symptoms,. Or an amount of a compound, ADC, or composition (eg, a harboxydiensplying regulator or ADC) described herein sufficient to produce a therapeutic effect, such as one that exhibits some other therapeutic efficacy. Point to. The effective amount can be determined by the method of the routine associated with the stated purpose. The term "therapeutically effective amount" refers to the detectable killing, reduction, and reduction of tumor cell growth or spread, tumor size or number, and / or other measures of cancer level, stage, progression and / or severity. / Or refers to the amount of compound, ADC, or composition described herein that is effective for inhibition. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the subject and disease condition under treatment, such as the weight and age of the subject, the severity of the disease condition, the method of administration, and the like. The vendor can easily determine. The term also applies to doses that will induce a particular response, eg, inhibition of cell growth, in a target cell. Specific doses are, for example, the detailed pharmaceutical composition, the subject and its age and risk of existing health or health status, the dosing regimen to be followed, the severity of the disease, which is administered in combination with other agents. It can vary depending on whether it is administered, the timing of administration, the tissue to which it is administered, and the physical delivery system to which it is carried. In the case of cancer, a therapeutically effective amount of ADC reduces the number of cancer cells, reduces tumor size, inhibits tumor metastasis (eg, slows or stops), and inhibits tumor growth (eg, slows or stops). And / or one or more symptoms can be alleviated.

"Prophylactically effective amount" refers to an amount effective at the dosage and duration required to achieve the desired prophylactic result. Typically, the prophylactic dose will be less than the therapeutically effective amount because the prophylactic dose is used in the subject prior to the disease or in the early stages of the disease.

As used herein, the terms "treating" or "therapeutic" and grammatically related terms result in prolonged survival, low morbidity, and / or reduction of side effects resulting from alternative therapeutic modality. Refers to any improvement in any consequences of the disease, such as. As is readily understood in the art, complete eradication of the disease is included, but not essential for therapeutic practice. "Treatment" or "treat" as used herein refers to the administration of the described ADC or composition to a subject, eg, a patient. Treatment is to cure, heal, alleviate, alleviate, change, improve, ameliorate, relieve, for example, disorders such as cancer, symptoms of the disorder or predisposition to the disorder. It can be improved or influenced by it. In some embodiments, in addition to treating a subject having a condition, the compositions disclosed herein are also provided prophylactically to prevent or reduce the likelihood of developing that condition. You can also do it.

In some embodiments, labeled ADCs are used. Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemically luminescent moieties, magnetic particles and the like.

As used herein, "protein" means at least two covalently linked amino acids. The term includes polypeptides, oligopeptides, and peptides. In some embodiments, two or more covalently linked amino acids are linked by peptide bonds. For example, when a protein is recombinantly made using an expression system and a host cell, the protein can be composed of naturally occurring amino acid and peptide bonds. Alternatively, the protein may contain synthetic amino acids (eg, homophenylalanine, citrulline, ornithine, and norleucine), or peptide mimetic structures, i.e., "peptides or protein analogs" such as peptoids. Peptoids are an exemplary peptide mimetics class whose side chains are attached to the nitrogen atom of the peptide backbone rather than the alpha carbon (as in the case of amino acids), with different hydrogen bonds and cons compared to the peptide. It has homemade characteristics (see, eg, Simon et al. (1992) Proc Natl Acad Sci. USA 89: 9637). As such, peptoids are resistant to proteolysis or other physiological or storage conditions and may be effective for cell membrane permeation. Such synthetic amino acids may be incorporated by conventional methods well known in the art, especially during in vitro synthesis of antibodies. In addition, any combination of peptide mimetics, synthetic and naturally occurring residues / structures can be used. "Amino acids" also include imino acid residues such as proline and hydroxyproline. The amino acid "R group" or "side chain" may be in either the (L) or (S) configuration. In a specific embodiment, the amino acids are in the (L) or (S) configuration.

A "recombinant protein" is a protein made using recombinant techniques using any technique and method known in the art, i.e. through expression of recombinant nucleic acids. Methods and techniques for producing recombinant proteins are well known in the art.

An "isolated" protein is, for example, at least a portion of the material normally associated with it in its natural state, which accounts for at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is not accompanied by. It is understood that in some circumstances, the isolated protein can account for 5% to 99.9% by weight of the total protein content. For example, an inducible promoter or a high expression promoter may be used to significantly increase the concentration of the protein so that the concentration level of the protein is increased. This definition includes the production of antibodies in a wide variety of organisms and / or host cells known in the art.

For amino acid sequences, sequence identity and / or similarity is not limited, but Smith and Waterman (1981) Adv Appl Math. 2: 482 Local Sequence Identity Algorithm, Needleman and Wunsch (1970) J Mol Biol. 48: 443 Sequence Identity Alignment Algorithm, Pearson and Lipman (1988) Proc Nat Acad Sci. USA 85: 2444 similarity search methods, computerized implementations of these algorithms (Wisconsin Computers Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. al. (1984) Nucl Acid Res. The Best Fit sequence program described by 12: 387-95, preferably using default settings, or may be determined using standard techniques known in the art, including by visual inspection. .. Preferably, the percent identity is calculated by FastDB based on the following parameters: mismatch penalty 1; gap penalty 1; gap size penalty 0.33; and joining penalty 30 (“Curent Methods in Sequence Comparison and Analysis,”. Macromolecule Analysis and Synchronizations, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.).

An example of a useful algorithm is PILEUP. PILEUP uses a gradual pairwise alignment to create a multiple sequence alignment from a group of related sequences. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP is available from Feng & Dolottle (1987) J Mol Evol. A simplification of the gradual alignment method of 35: 351-60 is used; this method is similar to that described by Higgins and Sharp (1989) CABIOS 5: 151-3. Useful PILEUP parameters including default gap weighting 3.00, default gap length weighting 0.10, and weighted end gaps.

Another example of a useful algorithm is Altschul et al. (1990) J Mol Biol. 215: 403-10; Altschul et al. (1997) Nucl Acid Res. 25: 3389-402; and Karin et al. (1993) Proc Natl Acad Sci. USA 90: 5873-87 is a BLAST algorithm described in. A particularly useful BLAST program is the WU-BLAST-2 program, which is described by Altschul et al. (1996) It was obtained from Methods in Enzyme 266: 460-80. WU-BLAST-2 uses several search parameters, most of which are set to default values. The adjustable parameters are set using the following values: overlap width = l, overlap rate = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the search for the sequence of interest is being performed; however, the sensitivity. These values may be adjusted to increase.

Further useful algorithms are described in Altschul et al. (1997) Nucl Acid Res. BLAST with a gap as reported by 25: 3389-402. BLAST with gaps is a BLASTUM-62 substitution score; a threshold T parameter set to 9; initiation of gapless elongation by the two-hit method, imposing a cost of 10 + k on the gap length of k; Xu set to 16 and. Use Xg set to 40 for the database search stage and 67 for the output stage of the algorithm. Gap alignment starts with a score corresponding to about 22 bits.

In general, the proteins and variants thereof disclosed herein, such as target antigens (HER2, CD138, or EPHA2, MSLN, SOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6. , SLC44A4, or STEAP1) and amino acid homology, similarity, or identity between variants of the antibody variable domain (including individual variant CDRs) is at least the sequence shown herein. 80%, eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, nearly 100%, or 100% homologity. Gender or identity.

Similarly, the "% (%) nucleic acid sequence identity" with respect to the nucleic acid sequences of the antibodies and other proteins identified herein is in a candidate sequence that is identical to a nucleotide residue in the coding sequence of an antigen-binding protein. Defined as the percentage of nucleotide residues in. As a specific method, the BLASTN module of WU-BLAST-2, in which the overlap width and the overlap rate are set to 1 and 0.125, respectively, set as the default parameters is used.

The site or region into which the amino acid sequence mutation is introduced is predetermined, but the mutation itself does not have to be predetermined. For example, random mutagenesis at a target codon or region to optimize mutation performance at a given site may be performed to screen the expressed antigen-binding protein CDR variants for the optimal combination of desired activity. good. Techniques for creating substitution mutations at predetermined sites in DNA with known sequences are well known, such as MI3 primer mutagenesis and PCR mutagenesis.

As used herein, "alkyl" or "alkyl group" means a fully saturated linear, branched, or cyclic hydrocarbon chain. In certain embodiments, the alkyl group may contain 1 to 8 carbon atoms (“C ₁ to C ₈ alkyl”). In certain embodiments, the alkyl group may contain 1 to 6 carbon atoms (“C ₁ to C ₆ alkyl”). In certain embodiments, the alkyl group contains 1-3 carbon atoms. Still in other embodiments, the alkyl group contains 2-3 carbon atoms, yet in other embodiments the alkyl group contains 1-2 carbon atoms.

"Alkoxy alkoxy" as used herein means an alkyl group substituted with an alkoxy group.

As used herein, "alkoxy" refers to an alkyl group as defined above, which is attached to the carbon backbone by an oxygen ("alkoxy") atom.

As used herein, "alkyl hydroxy" means an alkyl group substituted with a hydroxyl group.

"Hydroxy" or "hydroxyl" as used herein refers to -OH.

The "carbon ring" or "carbocyclyl", as used herein, comprises both aromatic (eg, aryl) and non-aromatic (eg, cycloalkyl) groups. In certain embodiments, the carbocyclic group contains 3-10 carbon atoms (“3-10-membered carbocycle”). In certain embodiments, the carbocyclic group contains 3-8 carbon atoms (“3-8 member carbocycles”). In certain embodiments, the carbocyclic group contains 3-6 carbon atoms (“3-6 member carbocycles”). In certain embodiments, the carbon ring group contains 3-5 carbon atoms (“3-5 membered carbon ring”).

"Haloalkyl" as used herein refers to an alkyl group substituted with one or more halogen atoms.

"Halogen" refers to any halogen group, such as -F, -Cl, -Br, or -I.

The terms "heterocycle", "heterocyclyl", and "heterocyclic", as used herein, are monocyclic heterocycles, bicyclic heterocycles, which contain at least one heteroatom in the ring. Or it means a tricyclic heterocycle.

A monocyclic heterocycle is a 3, 4, 5, 6, 7, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. In some embodiments, the heterocycle is a 3- or 4-membered ring containing one heteroatom selected from O, N and S. In some embodiments, the heterocycle is a 5-membered ring containing 0 or 1 double bond and 1, 2 or 3 heteroatoms selected from O, N and S. In some embodiments, the heterocycle contains 0, 1 or 2 double bonds and 6, 7 or 6 heteroatoms selected from O, N and S. It is an 8-membered ring. Typical examples of monocyclic heterocycles are, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxoranyl, dihydropyranyl (3,4-dihydro-2H-). (Including pyran-6-yl), 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isooxazolinyl, isooxazolidinyl, morpholinyl, oxadiazolinyl, Oxaziazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolinyl, thiadiazoline Examples thereof include lysynyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxide thiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.

The bicyclic heterocycles of the present disclosure include monocyclic heterocycles fused with an aryl group having a total of 5 to 12 ring atoms, monocyclic heterocycles fused with monocyclic cycloalkyl, or monocycles. A monocyclic heterocycle fused with the formula cycloalkenyl or a monocyclic heterocycle fused with the monocyclic heterocycle may be included. Examples of bicyclic heterocycles are, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-1,4-. Benzodioxynyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indrill, and 1,2,3,4-tetrahydroquinolinyl Can be mentioned.

The terms "heterocycle", "heterocyclyl", and "heterocyclic" include heteroaryls. "Heteroaryl" refers to a cyclic moiety having one or more ring closures, wherein at least one of the rings has one or more heteroatoms (oxygen, nitrogen or sulfur), wherein the ring. At least one of them is aromatic and one or more rings may be independently fused and / or crosslinked. Examples include, without limitation, phenyl, thiophenyl, triazolyl, pyridinyl, pyrimidinyl, pyridadinyl, and pyrazinyl.

As described herein, the compounds of the present disclosure may include "optionally substituted" moieties. In general, the term "substituted" means that one or more hydrogens in a specified portion are replaced with a suitable substituent, whether or not the term "optionally" precedes it. .. Unless otherwise indicated, a group "optionally substituted" may have a suitable substituent at each substitutable position of the group, with more than one position in any given structure. However, when it may be substituted with two or more substituents selected from the specified groups, the substituents may be the same or different at each position. The combinations of substituents envisioned under the present disclosure preferably result in the formation of stable compounds or chemically feasible compounds.

Those skilled in the art are subject to the permissible valences of the atoms and substituents to which such substitutions or absences are substituted, "substituted" or "substituted with" or "absent", and the substitutions or It will be appreciated that the absence results in the implicit condition that stable compounds are produced, such as compounds that do not spontaneously undergo conversion, such as by rearrangement, cyclization, elimination, etc. For the purposes of the present disclosure, a heteroatom such as nitrogen may have a hydrogen substituent and / or any acceptable substituent of the organic compound described herein that satisfies the valence of the heteroatom thereof.

"Stable" is also subject to conditions that allow its generation, detection, and, in certain embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein. Refers to a compound that does not change substantially chemically and / or physically. In some embodiments, stable or chemically feasible compounds are substantially unchanged when left at temperatures below 40 ° C. in the absence of moisture or under other chemical reaction conditions for at least one week. Is. In some embodiments, the compounds disclosed herein are stable.

The enantiomers taught herein include substantially a single enantiomer in a particular one or more asymmetric centers, eg, 90%, 92%, 95%, 98%, or 99% or more. Or "enantio pure" isomers containing a single enantiomer equal to 100% can be included. "Asymmetric center" or "chiral center" refers to a tetrahedral carbon atom containing four different substituents.

The compounds described herein may also contain unnatural proportions of isotopic atoms in one or more of the atoms constituting such a compound. For example, the compound may be radiolabeled with a radioisotope such as, for example, deuterium ( ² H), tritium ( ³ H), carbon 13 ( ¹³ C), or carbon 14 ( ¹⁴ C). All isotopic variants of the compounds disclosed herein are intended to be included within the scope of the present disclosure, whether radioactive or not. In addition, all tautomeric forms of the compounds described herein are intended to be within the claims.

Antibodies-Drug Conjugates The antibody-drug conjugates (ADCs) compounds of the present disclosure include those having anti-cancer activity. In particular, the ADC compound comprises an antibody or antigen binding fragment (including its antigen binding fragment) conjugated (ie, covalently linked by a linker) to a harboxydiene splicing regulator, eg, herein. Harboxydiene splicing regulators have cytotoxic or antiproliferative effects when not conjugated to an antibody or antigen binding fragment. In various embodiments, the harboxydiene splicing regulator has the ability to bind to and / or interact with the SF3b spliceosome complex when not conjugated to an antibody or antigen binding fragment. In various embodiments, the harboxydiene splicing regulator has the ability to regulate in vitro and / or in vivo RNA splicing when not conjugated to an antibody or antigen binding fragment. By targeting RNA splicing, in various embodiments, the harboxydiene splicing regulators and ADCs disclosed herein are potent antiproliferative agents. In various embodiments, the harboxydiene splicing regulators and ADCs disclosed herein can target both actively dividing and resting cells.

In various embodiments, the present disclosure is based, at least in part, on the finding that certain biologically active herboxydiene splicing regulators can provide improved properties when used in ADCs. Harboxydiene splicing regulators may exhibit desirable and improved characteristics when used alone (eg, robust SF3b spliceosome complex binding, strong RNA splicing regulatory), but in various embodiments, harboxy. Diene splicing regulators can exhibit the same desirable and improved feature reduction when conjugated to an antibody or antigen binding fragment. Thus, the development and production of human therapeutic agents, eg, ADCs for use as oncological agents, will be linked to a drug that binds to one or more desired targets and is used alone in the treatment of cancer. More than identifying capable antibodies may be needed. Linking an antibody to a harboxydiene splicing regulator can have a significant effect on the activity of one or both of the antibody and the harboxydien splicing regulator, which effect is the linker and / or harboxydien splicing of choice. It will vary depending on the type of regulator. Thus, in some embodiments, the components of the ADC are (ii) an antibody or antigen-binding fragment that retains one or more therapeutic properties that the antibody and herboxydienspiring regulatory moiety alone exhibit. Specific binding properties are maintained; (iii) harboxydiensplicing regulator loading and harboxydienplying regulator vs. antibody ratio are optimized; (iv) by stable binding to the antibody or antigen binding fragment. Delivery of the herboxydienspiring regulatory moiety, eg, intracellular delivery, is possible; (v) the stability of the ADC is maintained as an intact conjugate until transported or delivered to the target site; (vi) administration. Pre- or post-administration ADC aggregation is minimized; (vii) a therapeutic effect, eg, a cytotoxic effect, of the herboxydienspiring regulatory moiety is achieved after cleavage or other release mechanisms in the cellular environment; (viii). It exhibits in vivo anti-cancer therapeutic efficacy equal to or better than that of the antibody and herboxydiensplicing regulatory moiety alone; (ix) minimal off-target killing by the herboxydiensplicing regulatory moiety; and / or (X) Selected to exhibit the desired pharmacokinetic and medicinal properties, medicinability, and toxicological / immunological profile. Each of these properties may be required for the identification of improved ADCs for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14: 1605-13).

In various embodiments, the ADCs disclosed herein unexpectedly exhibit advantageous properties in some or each of the categories listed above. For example, in some embodiments, the ADC construct disclosed herein is surprisingly compared to an ADC containing another linker and / or drug moiety (eg, another herboxydiene splicing regulator). When off, while exhibiting a favorable harboxydiene splicing regulator loading, aggregation, and / or stability profile and / or maintaining antibody binding function, drug activity, and / or improved bystander killing. Target killing is reduced. In some embodiments, the ADC constructs disclosed herein have superior stability, activity, efficacy, or superior stability, activity, efficacy, or when compared to ADCs that use different linkers and / or harboxydiene splicing regulatory moieties. Demonstrate other effects (measured in vivo or in vitro). In some embodiments, the ADC constructs disclosed herein exhibit in vivo therapeutic efficacy when administered as a single dose. In some embodiments, the ADC constructs disclosed herein are surprisingly stable when compared to ADCs that use different linkers and / or herboxydiene splicing regulator moieties.

The ADC compounds of the present disclosure may selectively deliver effective doses of cytotoxic or cell proliferation inhibitors to cancer cells or tumor tissues. The disclosed ADCs express their respective target antigens (eg, HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEP1). It was discovered that it has a strong cytotoxic activity and / or a cell proliferation inhibitory activity against cells. In some embodiments, the cytotoxic and / or cell proliferation inhibitory activity of the ADC depends on the expression of the target antigen of the cell. In some embodiments, the disclosed ADCs are particularly effective in killing cancer cells expressing the target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and / or cell proliferation inhibitory effects on cancer cells that do not express the target antigen.

Exemplary cancers that express HER2 include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urinary epithelial cell cancer, esophageal cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous endometrial). Cancer), salivary tract cancer, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diamond Ther. 17: 85-99).

Exemplary cancers that express CD138 include, but are not limited to, intrathoracic cancer (eg, lung cancer, mesotheloma), skin cancer (eg, basal cell cancer, squamous epithelial cancer), head and neck cancer (eg, laryngeal). Hypopharyngeal, nasopharyngeal), breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urinary epithelial cancer), hematological malignancies (eg, multiple bone marrow) Myeloma such as tumor, B-cell malignant tumor, Hodgkin's lymphoma), and thyroid cancer (Szatmari et al. (2015) Dis Markers 2015: 796052).

Exemplary cancers expressing EPHA2 include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al. (2011)). Expert Opin The Targets 15 (1): 31-51).

In some embodiments, cleavage of the ADC releases the harboxydiene splicing regulator from the antibody or antigen binding fragment and linker. In some embodiments, the linker and / or harboxydiene splicing regulator is designed to promote bystander killing (killing of adjacent cells). In some embodiments, the linker and / or harboxydiene splicing regulator is cleaved after cell internalization and to adjacent cells the linker-harboxydiene splicing regulator moiety and / or the harboxidien splicing regulator moiety. Designed to promote bystander killing through a single spread. In some embodiments, the linker enhances cell internalization. In some embodiments, the linker minimizes cleavage in the extracellular environment, thereby reducing toxicity to off-target tissue (eg, non-cancerous tissue), while ADC binding to the target tissue, and It is designed to not express the antigen targeted by the antibody or antigen-binding fragment of the ADC, but to maintain bystander killing of the cancerous tissue surrounding the target cancer tissue expressing that antigen. In some embodiments, the catabolic product of the harboxydiene splicing regulatory moiety, or the harboxydiene splicing regulatory moiety produced by cleavage of the ADC, is uptake (ie, cell permeability) by the target cell or adjacent cells. Designed to promote. Such herboxydiene splicing regulatory moieties and catabolic products may be referred to herein as "bystander activity", while drug moieties or catabolic products with reduced cell permeability are "bystander inactive". May be referred to as.

In some embodiments, the disclosed ADCs also demonstrate bystander killing activity, but have low off-target cytotoxicity. Although not constrained by theory, ADC's bystander killing activity is particularly limited when its permeability to solid tumors and / or when target antigen expression is heterogeneous among tumor cells. Can be beneficial. In some embodiments, ADCs containing cleavable linkers are particularly effective in bystander killing compared to equivalent treatment with ADCs containing non-cleavable linkers, and / or demonstrate improved bystander killing activity. .. In some embodiments, the ADCs disclosed herein exhibit improved solubility and target cell permeability as compared to the drug moiety itself. In some embodiments, the ADCs disclosed herein exhibit improved cytotoxicity as compared to the harboxydiene splicing regulator moiety itself. In some embodiments, the ADC disclosed herein uses a drug moiety that exhibits low cytotoxicity when evaluated as a harboxidiene splicing regulator alone, but surprisingly alone. Is better than ADCs containing other cytotoxic splicing regulator moieties that have high cytotoxicity when evaluated as a herboxydiene splicing regulator. In some embodiments, cleavage and release of the harboxydiene splicing regulator enhances the cytotoxicity of the ADC as compared to equivalent treatment with an ADC containing a non-cleavable linker. In other embodiments, cleavage and release of the harboxydiene splicing regulator is not essential for the ADC to have the desired biological activity. In some embodiments, the ADC containing the non-cleavable linker (eg, ADL12) with increased spacer length is the same or the same as the equivalent treatment with the ADC containing the cleavable linker (eg, ADL1, ADL5). It results in a degree of cytotoxicity and, surprisingly, superior cytotoxicity compared to equivalent treatment with ADCs containing shorter non-cleavable linkers. In some embodiments, an ADC containing a non-cleavable linker (eg, ADL12) with an increased spacer length without a carbonyl group is equivalent to treatment with an ADC containing a cleavable linker (eg, ADL1, ADL5). Compared to equivalent treatment with an ADC containing a non-cleavable linker (eg, ADL10) that results in the same or similar degree of cytotoxicity as compared to and surprisingly has the same or similar spacer length containing a carbonyl group. Brings excellent cytotoxicity. In some embodiments, removing the carbonyl group from the uncleavable MC linker (eg, ADL12) is 50-fold compared to equivalent treatment with an ADC containing an unmodified non-cleavable MC linker (eg, ADL10). Greater, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold increase in cytotoxicity can occur. In some embodiments, removing the carbonyl group from the uncleavable MC linker (eg, ADL12) and increasing the spacer length (eg, addition of at least one spacer unit) results in unmodified uncleavable MC. An increase in cytotoxicity can occur greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold compared to equivalent treatment with ADCs containing a linker (eg, ADL10).

The present specification includes an antibody that targets tumor cells or an antigen-binding fragment thereof (Ab), a splicing regulator drug moiety (D), and a linker moiety (L) that covalently binds Ab to D. ADC compounds are provided. In certain embodiments, the antibody or antigen binding fragment is a tumor-related antigen (eg, HER2, CD138, EPHA2, MSLN, FULLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, Alternatively, it can bind to STEAP1) with high specificity and high affinity. In certain embodiments, the antibody or antigen binding fragment is incorporated into the target cell upon binding, eg, into a degradable compartment in the cell. In various embodiments, ADCs may be used that internalize on binding to target cells, cause degradation, release the harboxydien splicing regulatory moiety and kill the cancer cells. The harboxydiene splicing regulator moiety may be released from the antibody and / or linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

An exemplary ADC is the formula (I) :.
Ab- (L-H) _p (I)
(In the formula, Ab = antibody or antigen-binding fragment, L = linker moiety, H = harboxydiene splicing regulatory drug moiety, and p = number of splicing regulator drug moieties per antibody or antigen-binding fragment).

In certain embodiments, the harboxydiene splicing regulator targeting moiety for use in the described ADCs and compositions is an antibody or antigen binding fragment. Other exemplary drug targeting moieties for use in the described ADCs and compositions are also provided and described herein. In some embodiments, the harboxydiene splicing regulator targeting moiety may be any one of a variety of cell binders and non-antibody scaffolds. In some embodiments, the targeting moiety of the harboxydiene splicing regulator is a cell binding agent. As used herein, the term "cell binding agent" binds to animal (eg, human) cells and is a harboxydiene splicing regulatory moiety (eg, harboxy as disclosed herein). The diene splicing regulator refers to any drug capable of delivering the drug portion). The term includes exemplary antibodies and antigen-binding fragments disclosed herein (eg, monoclonal antibodies and fragments thereof such as Fab and scFV). The term further refers to exemplary cell binding agents such as DARPin, duobody, dicyclic peptide, nanobody, sentinyl, MSH (melanocyte stimulating hormone), receptor-Fc fusion molecule, T cell receptor structure, and the like. Includes steroid hormones such as androgen and estrogen, growth factors, colony stimulating factors such as EGF, and other non-antibody scaffolds. In various embodiments, the non-antibody scaffold can be broadly divided into two structural classes: domain-sized compounds (about 6-20 kDa) and constrained peptides (about 2-4 kDa). Exemplary domain-sized scaffolds include, but are not limited to, affibody, affiliin, proteinin, trimer, DARPin, FN3 scaffolds (eg, adnectin and sentinline), finomers. (Finomer), Knitz domain, pronectin, O-body, and receptor-Fc fusion proteins, while exemplary constrained peptides include avimer, bicyclic. Examples include peptides and Cys knots. In some embodiments, the drug targeting moieties used in the described ADCs and compositions are Affibody, Affiliin, Anticalin, Atrimer, DARPin, Adnectin or Sentiline. FN3 scaffolds such as, finomer, Knitz domain, pronectin, O-body, avimer, bicyclic peptide, and Cys knots. In some embodiments, the drug targeting moiety used in the described ADCs and compositions is a receptor-Fc fusion protein, such as a HER2-Fc chimeric fusion protein. For non-antibody scaffolds, see, for example, Vazquez-Lombardi et al. (2015) Review by Drug Dis Today 20 (10): 1271-83.

The antibody or antigen-binding fragment (Ab) of the antibody formula (I) includes, within its scope, any antibody or antigen-binding fragment that specifically binds to the target antigen on the cancer cell. The antibody or antigen binding fragment may bind to the target antigen with a dissociation constant (KD) of any size between ≤1 mM, _≤100 nM or ≤10 nM, as measured, for example, by BIAcore® analysis. In certain embodiments, the _KD is 1 pM to 500 pM. In some embodiments, _KD is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

In some embodiments, the antibody or antigen binding fragment is a four-stranded antibody (also referred to as an immunoglobulin or full-length antibody or intact antibody) and comprises two heavy chains and two light chains. .. In some embodiments, the antibody or antigen-binding fragment is a double-stranded half body (one light chain and one heavy chain), or an antigen-binding fragment of an immunoglobulin. In some embodiments, an antibody or antigen-binding fragment is an immunoglobulin antigen-binding fragment that retains the ability to bind to a target cancer antigen and / or provide the function of the immunoglobulin.

In some embodiments, the antibody or antigen binding fragment is an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment thereof. In some embodiments, the internalizing antibody or the internalizing antigen-binding fragment thereof binds to a target cancer antigen expressed on the surface of the cell and invades the cell upon binding. In some embodiments, the drug portion of the harboxydienspiring regulator of the ADC is after the ADC has invaded and is present in the cells expressing the target cancer antigen (ie, the ADC has been internalized). After), for example, by cleavage, degradation of the antibody or antigen-binding fragment, or by any other suitable release mechanism, it is released from the antibody or antigen-binding fragment of the ADC.

The amino acid sequences of the exemplary antibodies of the present disclosure are shown in Tables 2-4.

In various embodiments, the ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the table above, or one from a set of heavy and light chains. A set of 6 CDR sequences may be included, for example, by transplanting those 6 CDRs into a human donor antibody framework of choice. In various embodiments, the ADC disclosed herein retains the ability of the ADC to bind to its target cancer antigen (eg, with a KD of less than 1 × ^10-8 _M ) and is described herein. With the sequences listed in the table above, as long as they retain one or more of the disclosed functional properties of the ADC (eg, the ability to internalize, regulate RNA splicing, inhibit cell growth, etc.). It may contain homologous amino acid sequences.

In some embodiments, the ADC further comprises a human heavy and light chain constant domain or a fragment thereof. For example, the ADC may include a human IgG heavy chain constant domain (such as IgG1) and a human kappa or lambda light chain constant domain. In various embodiments, the antibody or antigen binding fragment of the described ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain along with a human Ig kappa light chain constant domain.

In various other embodiments, the target cancer antigen of the ADC is human epidermal growth factor receptor 2 (HER2).

In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is heavy chain CDR1 consisting of SEQ ID NO: 1 as defined by the following three heavy chain CDRs and three light chain CDRs: Kabat numbering scheme: HCDR1), heavy chain CDR2 consisting of SEQ ID NO: 2 (HCDR2), heavy chain CDR3 consisting of SEQ ID NO: 3 (HCDR3); light chain CDR1 consisting of SEQ ID NO: 4 (LCDR1), light chain CDR2 consisting of SEQ ID NO: 5 (LCDR2). , And a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 6.

In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 19 and the light chain variable region amino acid sequence of SEQ ID NO: 20, or the disclosed sequence. Contains an array. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19 heavy chain variable region amino acid sequence and / or SEQ ID NO. It has a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to 20.

In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-HER2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

In various embodiments, the anti-HER2 antibody is at least 95% identical to the heavy chain amino acid sequence or SEQ ID NO: 19 of SEQ ID NO: 19 and at least 95% identical to the light chain amino acid sequence or SEQ ID NO: 20 of SEQ ID NO: 20. Contains an array. In a detailed embodiment, the anti-HER2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 19 and a light chain amino acid sequence of SEQ ID NO: 20, or at least 95% identical to the disclosed sequence. In some embodiments, the anti-HER2 antibody is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19 with the same heavy chain amino acid sequence and at least 96%, at least 97% with SEQ ID NO: 20. , At least 98%, or at least 99% of the same light chain amino acid sequence. In various embodiments, the anti-HER2 antibody is trastuzumab, or an antigen-binding fragment thereof.

In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of trussumab, where the CDRs are HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2). , HCDR3 (SEQ ID NO: 3); 1, 2, 3, 4, 5, or 6 amino acid additions, deletions or substitutions of LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5), and LCDR3 (SEQ ID NO: 6). Contains less than one.

In various other embodiments, the target cancer antigen of the ADC is human syndecane-1 (CD138).

In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is heavy chain CDR1 consisting of SEQ ID NO: 7, as defined by the following three heavy chain CDRs and three light chain CDRs: Kabat numbering scheme: HCDR1), heavy chain CDR2 consisting of SEQ ID NO: 8 (HCDR2), heavy chain CDR3 consisting of SEQ ID NO: 9 (HCDR3); light chain CDR1 consisting of SEQ ID NO: 10 (LCDR1), light chain CDR2 consisting of SEQ ID NO: 11 (LCDR2). , And a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 12.

In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 21 and the light chain variable region amino acid sequence of SEQ ID NO: 22 or the disclosed sequence. Contains an array. In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 heavy chain variable region amino acid sequence and / or SEQ ID NO. It has a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to 22.

In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-CD138 antibody comprises a mouse IgG2a heavy chain constant domain and a mouse Ig kappa light chain constant domain. In various embodiments, the anti-CD138 antibody comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain.

In various embodiments, the anti-CD138 antibody is at least 95% identical to the heavy chain amino acid sequence or SEQ ID NO: 21 of SEQ ID NO: 21 and at least 95% identical to the light chain amino acid sequence or SEQ ID NO: 22 of SEQ ID NO: 22. Contains an array. In a detailed embodiment, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22, or at least 95% identical to the disclosed sequence. In some embodiments, the anti-CD138 antibody is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 and at least 96%, at least 97% identical to SEQ ID NO: 22. , At least 98%, or at least 99% have the same light chain amino acid sequence. In various embodiments, the anti-CD138 antibody is B-B4, or an antigen-binding fragment thereof.

In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of B-B4, wherein the CDRs are HCDR1 (SEQ ID NO: 7), HCDR2 (SEQ ID NO: 7). 8), HCDR3 (SEQ ID NO: 9); 1, 2, 3, 4, 5, for amino acid additions, deletions or substitutions of LCDR1 (SEQ ID NO: 10), LCDR2 (SEQ ID NO: 11), and LCDR3 (SEQ ID NO: 12). Or it contains less than 6 pieces.

In various other embodiments, the target cancer anticancer of the ADC is human effrin type A receptor 2 (EPHA2).

In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is heavy chain CDR1 consisting of SEQ ID NO: 13 as defined by the following three heavy chain CDRs and three light chain CDRs: Kabat numbering scheme: HCDR1), heavy chain CDR2 consisting of SEQ ID NO: 14 (HCDR2), heavy chain CDR3 consisting of SEQ ID NO: 15 (HCDR3); light chain CDR1 consisting of SEQ ID NO: 16 (LCDR1), light chain CDR2 consisting of SEQ ID NO: 17 (LCDR2). , And a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18.

In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 23 and the light chain variable region amino acid sequence of SEQ ID NO: 24, or the disclosed sequence. Contains an array. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23 heavy chain variable region amino acid sequence and / or SEQ ID NO. It has a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to 24.

In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-EPHA2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

In various embodiments, the anti-EPHA2 antibody is at least 95% identical to the heavy chain amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 23, and at least 95% identical to the light chain amino acid sequence or SEQ ID NO: 24 of SEQ ID NO: 24. Contains an array. In a detailed embodiment, the anti-EPHA2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 23 and a light chain amino acid sequence of SEQ ID NO: 24, or a sequence that is at least 95% identical to the disclosed sequence. In some embodiments, the anti-EPHA2 antibody is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23 and at least 96%, at least 97% identical to SEQ ID NO: 24. , At least 98%, or at least 99% have the same light chain amino acid sequence. In some embodiments, the anti-EPHA2 antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 23; and a light chain encoded by the nucleotide sequence of SEQ ID NO: 24. In various embodiments, the anti-EPHA2 antibody is 1C1 or an antigen-binding fragment thereof.

In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises 3 heavy chain CDRs and 3 light chain CDRs of 1C1, where the CDRs are HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14). , HCDR3 (SEQ ID NO: 15); 1, 2, 3, 4, 5, or 6 amino acid additions, deletions or substitutions of LCDR1 (SEQ ID NO: 16), LCDR2 (SEQ ID NO: 17), and LCDR3 (SEQ ID NO: 18). Contains less than one.

In various embodiments, the amino acid substitution is of a single residue. Insertions can typically be on the order of about 1 to about 20 amino acid residues, but significantly larger insertions may be tolerated as long as biological function (eg, binding to the target antigen) is retained. Deletions usually range from about 1 to about 20 amino acid residues, but in some cases the deletions may be much larger. Substitutions, deletions, insertions, or any combination thereof can be used to reach the final derivative or variant. In general, these changes are made to a small number of amino acids in order to minimize changes in the molecule, especially in the immunogenicity and specificity of the antigen-binding protein. However, in certain circumstances, larger changes may be tolerated. In general, conservative substitutions are made according to the chart below, shown in Table 6.

Substantial changes in function or immunological identity are made by choosing less conservative substitutions than those shown in Table 6. For example, substitutions may be made that have a more significant effect on the structure of the polypeptide backbone in the region of change, such as the α-helix or β-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chains. Substitutions that can generally cause the greatest changes in the properties of polypeptides are as follows: (a) Hydrophilic residues such as ceryl or threonyl replace hydrophobic residues such as leucyl, isoleucyl, phenylalanyl, valyl or alanyl. Substituted with (or thereby); (b) cysteine or proline is substituted (or thereby) in place of any other residue; (c) residue with an electrically positive side chain, eg. , Lysyl, arginyl, or histidyl is replaced (or thereby) with an electrically negative residue, such as glutamil or aspartyl; or (d) a residue with a bulky side chain, such as phenylalanine, is sided. Those that do not have chains, such as those that are replaced (or thereby) in place of glycine.

In various embodiments in which a variant antibody sequence is used for ADC, the variant will typically exhibit the same qualitative biological activity and elicit the same immune response, but the variant is also required. It may be selected to modify the characteristics of the antigen-binding protein accordingly. Alternatively, the variant may be designed to alter the biological activity of the antigen binding protein. For example, the glycosylation site may be altered or removed.

Various antibodies may be used with the ADCs used herein to target cancer cells. As shown below, the linker-payload of the ADC disclosed herein is surprisingly effective with a variety of tumor antigen targeting antibodies. Suitable antigens that are expressed in tumor cells but not in healthy cells or are highly expressed in tumor cells as compared to healthy cells are known in the art, as are antibodies against them. These antibodies may be used with the linker and harboxydiene splicing regulator payload disclosed herein. In some embodiments, the antibody or antigen binding fragment targets HER2 and the HER2 targeting antibody or antigen binding fragment is trastuzumab. In some embodiments, the antibody or antigen binding fragment targets CD138 and the CD138 targeting antibody or antigen binding fragment is BB4. In some embodiments, the antibody or antigen binding fragment targets EPHA2 and the EPHA2 targeting antibody or antigen binding fragment is 1C1. In some embodiments, the disclosed linker and harboxidien splicing regulator payloads are surprisingly effective with several different tumor targeting antibodies, such as HER2 targeting antibodies such as trastuzumab, BB4 and the like. CD138-targeted antibodies, and EPHA2-targeted antibodies such as 1C1 have particularly improved drug: antibody ratio, aggregation level, stability (ie, in vitro and in vivo stability), tumor targeting (ie, cytotoxicity, efficacy). ) And / or brought about therapeutic efficacy. Increased therapeutic efficacy can be measured in vitro or in vivo and may include decreased tumor growth rate and / or decreased tumor volume.

In certain embodiments, different antibodies against the same target or antibodies against different antigen targets are used and have the advantageous functional properties described above (eg, improved stability, improved tumor targeting, improved therapeutic efficacy). Etc.) bring at least part of it. In some embodiments, these advantageous functions when the disclosed linker and herboxydiene splicing regulator payloads are conjugated to another HER2-targeted, CD138-targeted, or EPHA2-targeted antibody or antigen-binding fragment. Some or all of the properties are observed. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and herboxydiene splicing regulator payloads are conjugated to a HER2-targeted antibody or antigen binding fragment. To. In some embodiments, the antibody or antigen binding fragment targets HER2. In some embodiments, the HER2 targeting antibody or antigen binding fragment is trastuzumab. In some other embodiments, when the disclosed linker and herboxydiene splicing regulator payloads are conjugated to a CD138 targeting antibody or antigen binding fragment, some or all of these advantageous functional properties are observed. To. In some embodiments, the antibody or antigen binding fragment targets CD138. In some embodiments, the CD138 targeting antibody or antigen binding fragment is BB4. In some other embodiments, when the disclosed linker and herboxydiene splicing regulator payloads are conjugated to EPHA2 targeted antibodies or antigen binding fragments, some or all of these advantageous functional properties are observed. To. In some embodiments, the antibody or antigen binding fragment targets EPHA2. In some embodiments, the EPHA2 targeting antibody or antigen binding fragment is 1C1.

Linkers In various embodiments, ADC linkers are extracellularly stable enough to be therapeutically effective. In some embodiments, the linker is stable outside the cell so that the ADC remains intact when present in extracellular conditions (eg, prior to transport or delivery to the cell). As used in the context of ADC, the term "intact" means that an antibody or antigen binding fragment remains bound to a drug moiety (eg, a harboxydiene splicing regulator). As used herein, "stable" is, in the context of a linker or ADC containing a linker, cleaves (or the entire ADC) of the linkers in the sample of the ADC when the ADC is present under extracellular conditions. 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less (or any percentage in between) Means that In some embodiments, the linkers and / or ADCs disclosed herein are surprisingly compared to an ADC having another linker and / or another linker and / or harboxydiene splicing regulator payload. And stable. In some embodiments, the ADC disclosed herein can remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.

Whether the linker is extracellularly stable is determined, for example, by including ADC in plasma for a predetermined time (eg, 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then in plasma. It can be determined by quantifying the amount of free drug moiety present in. Stability gives the ADC sufficient time to localize to the target tumor cells and premature release of the drug moiety, which is due to indiscriminate damage to both normal and tumor tissue. The treatment index may decrease). In some embodiments, the linker is stable outside the target cell, and upon entering the cell, releases the drug moiety from the ADC and the drug targets the target (eg, to the SF3b spliceosome complex). You will be able to combine. Thus, an effective linker maintains (i) the specific binding properties of the antibody or antigen-binding fragment; (ii) delivery of the drug moiety by stable binding to the antibody or antigen-binding fragment, eg, intracellular delivery. Enables; (iii) remains stable and antigenic until the ADC has been transported or delivered to its target site; and (iv) the therapeutic effect of the drug moiety, eg, cytotoxic effect, after cleavage or another release mechanism. Will be realized.

The linker can affect the physicochemical properties of the ADC. As many cytotoxic agents are hydrophobic in nature, ligation to antibodies with additional hydrophobic moieties can lead to aggregation. ADC aggregates are insoluble and often limit the achievable drug load on the antibody, which can adversely affect the efficacy of the ADC. Biopharmaceutical protein aggregates are also generally associated with increased immunogenicity. As shown below, the linkers disclosed herein provide ADCs with low aggregation levels and desirable drug loading levels.

The linker may be "cleavable" or "non-cleavable" (Ducry and Stamp (2010) Bioconjugate Chem. 21: 5-13). Cleavable linkers are designed to release drug moieties (eg, harboxydiene splicing regulators) when exposed to specific environmental factors, eg, internalized into target cells, while being non-cleavable. Linkers generally rely on the degradation of the antibody or antigen binding fragment itself.

In some embodiments, the linker is a non-cleavable linker. In some embodiments, the drug portion of the harboxydiene splicing regulator of the ADC is released by degradation of the antibody or antigen binding fragment. Non-cleavable linkers tend to remain covalently associated with at least one amino acid of an antibody and drug upon internalization by the target cell and degradation within it. A number of exemplary non-cleavable linkers are described herein and are also known in the art. Exemplary non-cleavable linkers are thioether, cyclohexyl, N-succinimidyl 4- (N-maleimidemethyl) cyclohexane-1carboxylate (SMCC), or N-hydroxysuccinimide (NHS), one or more polyethylene glycols (PEG). It may contain moieties such as 1, 2, 3, 4, 5, or 6 PEG moieties, or 1 or more alkyl moieties.

In some embodiments, the linker is a cleavable linker. The cleavable linker refers to any linker containing a cleavable moiety. As used herein, the term "cleaveable portion" refers to any chemical bond that can be cleaved. Suitable cleavable chemical bonds are well known in the art and include, but are not limited to, acid-unstable bonds, protease / peptidase-unstable bonds, photo-unstable bonds, disulfide bonds, and esterase-unstable bonds. A linker containing a cleavable moiety may allow the release of the harboxydiene splicing regulatory drug moiety from the ADC by cleavage at a particular site of the linker.

In some embodiments, the linker is cleavable under intracellular conditions, so that in the intracellular environment, cleavage of the linker is sufficient to release the harboxydiensplicing regulatory drug moiety from the antibody or antigen binding fragment. The drug is activated and / or the drug is therapeutically effective. In some embodiments, the drug portion of the harboxydiene splicing regulator is not cleaved from the antibody or antigen-binding fragment until the ADC enters cells expressing an antigen specific for the antibody or antigen-binding fragment of the ADC. Upon entering the cell, the drug portion of the harboxidiene splicing regulator is cleaved from the antibody or antigen binding fragment. In some embodiments, the linker comprises a cleavable moiety that is positioned such that no portion of the linker or antibody or antigen binding fragment remains bound to the harboxydiene splicing regulatory drug moiety upon cleavage. Exemplary cleavable linkers include acid unstable linkers, protease / peptidase sensitive linkers, photolabile linkers, dimethyl-containing, disulfide-containing, or sulfonamide-containing linkers.

In some embodiments, the linker is a pH sensitive linker and is sensitive to hydrolysis at a particular pH value. Typically, the pH sensitive linker can be cleaved under acidic conditions. This cleavage strategy takes advantage of the generally lower pH of the intracellular compartments of endosomes (pH about 5-6) and lysosomes (pH about 4.8) when compared to cytosol (pH about 7.4). , Hydrazone, etc., which cause hydrolysis of acid instability groups in the linker (Jin et al. (2015) Pharm Res 32: 3526-40). In some embodiments, the linker is an acid unstable and / or hydrolyzable linker. For example, an acid instability linker that is hydrolyzable within the lysosome and contains an acid instability group (eg, hydrazone, semicarbazone, thiosemicarbazone, cis-aconite acid amide, ortho ester, acetal, ketal, etc.). Can be used. For example, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker (1999) Pharma Therapys 83: 67-123; Neverle et al. (1989) Biol Chem. See 264: 14653-61. Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (eg, thioether linked to a therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929). ).

In some embodiments, the linker is cleaveable under reducing conditions. In some embodiments, the linker is cleavable in the presence of a reducing agent such as glutathione or dithiothreitol. In some embodiments, the linker is a cleavable disulfide linker or a cleavable sulfonamide linker.

In some embodiments, the linker is a cleavable disulfide linker. Various disulfide linkers include, for example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), SPDB (N-succinimidyl-3- (2-pyridyl)). Dithio) butyrate) and SMPT (N-succinimidyloxycarbonyl-α-methyl-α- (2-pyridyl-dithio) toluene), including those that can be formed using SPDB and SMPT. Known in the field. For example, Thorpe et al. (1987) Cancer Res. 47: 5924-31; Wawrzynczak et al. , In Immunoconjugates: Antibodies Conjugates in Radioimagery and Therapy of Cancer (CW Voguel ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935. Disulfide linkers are typically used taking advantage of the large amount of intracellular thiols that can facilitate the cleavage of their disulfide bonds. The intracellular concentration of the most abundant intracellular thiol, reduced glutathione, is generally in the range of 1-10 nM, which is about 1,000 of the most abundant small molecule thiols (ie, cysteine) at about 5 μM in the blood. Double height (Goldmacher et al., In Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (GL Phillips ed), S. L. Phillips ed. The intracellular enzymes of the protein disulfide isomerase family can also contribute to the intracellular cleavage of the disulfide linker. As used herein, a cleavable disulfide linker refers to any linker containing a cleavable disulfide moiety. The term "cleaveable disulfide moiety" refers to a disulfide bond that can be cleaved and / or reduced, for example by a thiol or enzyme.

In some embodiments, the linker is a cleavable sulfonamide linker. As used herein, cleavable sulfonamide linker refers to any linker that contains a cleavable sulfonamide moiety. The term "cleavable sulfonamide moiety" refers to a sulfonamide group, i.e. a sulfonyl group linked to an amine group, where the sulfur-nitrogen bond can be cleaved.

In some embodiments, the linker may be a dendritic linker for covalently linking two or more drug moieties to an antibody or antigen binding fragment via a branched polyfunctional linker moiety. For example, Sun et al. (2002) Bioorg Med Chem Let. 12: 2213-5; Sun et al. (2003) Bioorg Med Chem. 11: 1761-8. The dendritic linker can increase the drug-to-antibody molar ratio, i.e., the drug loading, which is related to the efficacy of the ADC. Thus, if the antibody or antigen binding fragment carries only one reactive cysteine thiol group, for example, a dendritic linker may bind multiple harboxydiene splicing regulatory drug moieties. In some embodiments, the linker moiety or linker-drug moiety may be linked to an antibody or antigen binding fragment by reduced disulfide cross-linking chemistry or lysine limited utilization techniques. See, for example, International Publication No. 2013/173391 and Pamphlet 2013/173393.

In some embodiments, the linker can be cleaved by a cleaving agent present in the intracellular environment (eg, in lysosomes or endosomes or caveolae), such as an enzyme. The linker may be, for example, an intracellular peptidase or a peptide linker cleaved by a protease enzyme including, but not limited to, lysosomal or endosomal proteases.

In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker containing a cleavable peptide moiety. The term "cleavable peptide moiety" refers to any chemically bound linked amino acid (natural or synthetic amino acid derivative) that can be cleaved by a drug present in the intracellular environment. For example, the linker may contain a valine-alanine (Val-Ala) sequence or a valine-citrulline (Val-Cit) sequence that can be cleaved by a cathepsin, such as cathepsin B. In some embodiments, the linker may comprise a glutamic acid-valine-citrulline sequence (Glu-Val-Cit). In some embodiments, the linker is an enzymatically cleavable linker and the cleavable peptide moiety in the linker is enzymatically cleavable. In some embodiments, the cleavable peptide moiety is cleavable by a lysosomal enzyme such as a cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the cleavable peptide moiety in the linker is lysosomal cysteine cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. .. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B. An exemplary dipeptide that can be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13: 855-69).

In some embodiments, the linker or the cleavable peptide moiety in the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows cleavage of the linker by a protease, thus a harboxydiene splicing regulator drug from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes. Promotes the release of moieties (Doronina et al. (2003) Nat Biotechnol. 21: 778-84; Dubowchik and Walker (1999) Pharma Therapys 83: 67-123). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-alanine (Val-Ala), valine-citrulin (Val-Cit), alanin-asparagin (Ala-Asn), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine. (Phe-Lys), alanine-lysine (Ala-Lys), alanine-valine (Ala-Val), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulin (Phe-Cit) , Leucine-citrulin (Leu-Cit), isoleucine-citrulin (Ile-Cit), tryptophan-citrulin (Trp-Cit), and phenylalanine-alanine (Phe-Ala). Exemplary tripeptides include, but are not limited to, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine-citrulin (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly). , Phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), glutamate-valine-citrulin (Glu-Val-Cit) (see, eg, Anami et al. (2018) Nat Com. 9: 2512), and glycine. -Phenylalanine-lysine (Gly-Phe-Lys) can be mentioned. Other exemplary amino acid units are, but are not limited to, Gly-Phe-Gly-Gly (SEQ ID NO: 34), Gly-Phe-, as described, for example, in US Pat. No. 6,214,345. Examples include Leu-Gly (SEQ ID NO: 35), Ala-Leu-Ala-Leu (SEQ ID NO: 36), Phe-N ⁹ -tosyl-Arg, and Phe-N ⁹ -nitro-Arg. In some embodiments, the amino acid unit in the linker comprises Val-Ala. In some embodiments, the amino acid unit in the linker comprises Val-Cit. In some embodiments, the amino acid unit in the linker comprises Glu-Val-Cit. Amino acid units can include naturally occurring amino acid residues and / or trace amino acids and / or non-naturally occurring amino acid analogs such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by specific enzymes such as tumor-related proteases, lysosomal proteases such as cathepsins B, C, D, or S, or plasmin proteases.

を含む。 In some embodiments, the linker is a cleavable β-glucuronide linker. As used herein, cleavable β-glucuronide linker refers to any linker that contains a cleavable β-glucuronide moiety. An exemplary cleavable β-glucuronide linker has a structure:

including.

The term "cleavable β-glucuronide moiety" refers to a glycosidic bond that can be cleaved by a drug with β-glucuronidase activity. In some embodiments, the linker comprises a glycosidic bond that can be cleaved by β-glucuronidase. β-Glucronidase is a UDP-glucuronosyltransferase that catalyzes the hydrolysis of glycosidic bonds of glucuronides having a β configuration.

In some embodiments, the ADC disclosed herein comprises a cleavable β-glucuronide moiety in the linker that is cleavable by the enzyme. In some embodiments, the cleavable β-glucuronide moiety in the linker is cleavable by a lysosomal enzyme such as β-glucuronidase. In some embodiments, the linker is a β-glucuronidase cleavable linker. In some embodiments, the cleavable β-glucuronide moiety in the linker allows cleavage of the linker by β-glucuronidase after the internalization of the ADC, thereby releasing the drug moiety from the ADC within the cellular environment. To promote.

In some embodiments, the linker in any ADC disclosed herein comprises at least one spacer unit that binds an antibody or antigen binding fragment to a drug moiety (eg, a harboxydiene splicing regulatory drug moiety). Can include. In some embodiments, the spacer unit between the antibody or antigen binding fragment and the cleavable moiety, if present, connects the cleavage site in the linker (eg, the cleavable peptide moiety) to the antibody or antigen binding fragment. match. In some embodiments, the spacer unit between the drug moiety and the cleavable moiety, if present, ligates the cleavage site in the linker (eg, the cleavable peptide moiety) to the drug moiety. In some embodiments, there are no cleavage sites and spacer units are used to ligate the antibody or antigen binding fragment to the drug moiety.

In some embodiments, the linker and / or the spacer unit in the linker is substantially hydrophilic. The use of hydrophilic linkers can reduce the extent to which a drug can be extruded from resistant cancer cells by multidrug resistance (MDR) or functionally similar transporters. In some embodiments, the hydrophilic linker may contain one or more polyethylene glycol (PEG) moieties, such as 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the linker comprises two PEG moieties.

In some embodiments, the spacer unit in the linker comprises one or more PEG moieties. In some embodiments, the spacer unit comprises one or more-(PEG) _m -where m is an integer of 1-10 (ie, m is 1, 2, 3, 4, 5, 6, ... It can be 7, 8, 9, or 10). In some embodiments, m ranges from 1 to 10; 2 to 8; 2 to 6; 2 to 5; 2 to 4; or 2 to 3. In some embodiments, m is 2. In some embodiments, the spacer units are (PEG) ₂ , (PEG) ₃ , (PEG) ₄ , (PEG) ₅ , (PEG) ₆ , (PEG) ₇ , (PEG) ₈ , (PEG) ₉ , Or (PEG) ₁₀ . In some embodiments, the spacer unit comprises (PEG) ₂ .

In some embodiments, the spacer unit in the linker comprises an alkyl moiety. In some embodiments, the spacer unit comprises one or more-(CH ₂ ) _n- , where n is an integer of 1-10 (ie, n is 1, 2, 3, 4, 5, 6). , 7, 8, 9, or 10). In some embodiments, n is in the range of 1-10; 2-8; 2-6; 2-5; 2-4; or 2-3. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, the spacer units are (CH ₂ ) ₂ , (CH ₂ ) ₃ , (CH ₂ ) ₄ , (CH ₂ ) ₅ , (CH ₂ ) ₆ , (CH ₂ ) ₇ , (CH ₂ ). ) ₈ , (CH ₂ ) ₉ , or (CH ₂ ) ₁₀ . In some embodiments, the spacer unit comprises (CH ₂ ) ₂ (“Et”). In some embodiments, the spacer unit comprises (CH ₂ ) ₆ (“Hex”). In some embodiments, the spacer unit comprises (CH ₂ ) ₂ -O- (CH ₂ ) ₂ (“Et—O—Et”).

Spacer units can be used, for example, to directly or indirectly link an antibody or antigen binding fragment to a drug moiety. In some embodiments, the spacer unit links the antibody or antigen binding fragment directly to the harboxydiene splicing regulatory drug moiety. In some embodiments, the antibody or antigen binding fragment and the harboxydiene splicing regulator drug moiety are one or more PEG moieties (eg, (PEG) ₂ ), or one or more alkyl moieties (eg, (eg,). It is linked via a spacer unit containing CH ₂ ) ₂ , (CH ₂ ) ₆ , or (CH ₂ ) ₂ -O- (CH ₂ ) ₂ ). In some embodiments, the spacer unit indirectly links the antibody or antigen binding fragment to the drug portion of the harboxidiene splicing regulator. In some embodiments, the spacer unit comprises an antibody or antigen binding fragment into the harboxidiene splicing regulator drug moiety, a cleavable moiety (eg, a cleavable peptide or cleavable β-glucuronide) and / or a spacer unit. It is indirectly linked through a binding moiety for binding to an antibody or antigen binding fragment, such as a maleimide moiety.

In various embodiments, the spacer unit binds to an antibody or antigen-binding fragment (ie, antibody or antigen-binding fragment) via a maleimide (Mal) moiety.

A spacer unit that binds to an antibody or antigen-binding fragment via Mal is referred to herein as a "Mal-spacer unit." The term "Mal" or "maleimide moiety", as used herein, contains a maleimide group and is reactive with a sulfhydryl group, eg, a sulfhydryl group of a cysteine residue on an antibody or antigen binding fragment. Means a compound. Other functional groups having reactivity with sulfhydryl groups (thiols) include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates. In some embodiments, the Mal-spacer unit is reactive with cysteine residues on the antibody or antigen binding fragment. In some embodiments, the Mal-spacer unit is conjugated to an antibody or antigen binding fragment via a cysteine residue. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises an alkyl moiety.

In certain embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the amino acid unit comprises Glu-Val-Cit. In some embodiments, the linker comprises a Mal-spacer unit and a Val-Cit. In some embodiments, the linker comprises a Mal-spacer unit and Val-Ala. In some embodiments, the linker comprises a Mal-spacer unit and a Val-Cit, where the Male-spacer unit comprises a maleimide caproyl (MC). In some embodiments, the linker comprises a Mal-spacer unit and a Val-Ala, where the Male-spacer unit comprises a maleimide caproyl (MC). In some embodiments, the linker comprises a Mal-spacer unit and a cleavable β-glucuronide moiety.

In some embodiments, the linker comprises a structure: Mal-spacer unit. In some embodiments, the Mal-spacer unit comprises maleimide caproyl (MC). In some embodiments, the linker comprises structure: MC. In some embodiments, the linker comprises structure: Mal- (CH ₂ ) ₂ (“Mal-Et”). In some embodiments, the linker comprises structure: Mal- (CH ₂ ) ₆ (“Mal-Hex”). In some embodiments, the linker comprises a structure: Mal- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ ("Mal-Et-O-Et"). In some embodiments, the linker comprises structure: Mal- (PEG) ₂ . In some embodiments, the linker comprises structure: Mal- (PEG) ₂ -CO.

In various embodiments, the Mal-spacer unit binds an antibody or antigen-binding fragment to a cleavable peptide moiety. In some embodiments, the linker comprises a Mal-spacer unit-peptide. In some embodiments, the linker comprises structure: Mal-spacer unit-Val-Cit. In some embodiments, the Mal-spacer unit comprises maleimide caproyl (MC). In some embodiments, the linker comprises structure: MC-Val-Cit.

In some embodiments, the linker comprises structure: Mal-spacer unit-Val-Ala. In some embodiments, the Mal-spacer unit comprises maleimide caproyl (MC). In some embodiments, the linker comprises structure: MC-Val-Ala.

In various embodiments, the Mal-spacer unit binds an antibody or antigen-binding fragment to a cleavable β-glucuronide moiety. In some embodiments, the linker comprises the Mal-spacer unit-β-glucuronide. In some embodiments, the linker comprises MC-β-glucuronide.

In various embodiments, the cleavable portion of the linker is directly tethered to the harboxydiene splicing regulator drug portion. In other embodiments, spacer units are used to tie the cleavable portion of the linker to the harboxydiene splicing regulator drug portion. In various embodiments, the harboxydiene splicing regulator is bound to the cleavable portion of the linker by a spacer unit.

The spacer unit may be "self-sacrificing" or "non-self-sacrificing". A "non-self-sacrificing" spacer unit is one in which some or all of the spacer unit remains bound to the drug portion of the harboxydiene splicing regulator upon cleavage of the linker. Examples of non-self-sacrificing spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Non-self-sacrificing spacer units can eventually degrade over time, but do not immediately release the entire linked undenatured drug moiety, even under cellular conditions. The "self-sacrificing" spacer unit allows the release of the undenatured drug moiety under intracellular conditions. A "non-denatured drug" or "undenatured drug moiety" is one in which no portion or other chemical modification of the spacer unit remains after cleavage / decomposition of the spacer unit.

Self-sacrificing chemistry is known in the art and can be readily selected for the disclosed ADCs. In various embodiments, the spacer unit that binds the cleavable portion of the linker to the harboxydiene splicing regulator drug moiety is self-sacrificing and self-sacrificing at the same time as or immediately before / immediately after cleavage of the cleavable moiety under intracellular conditions. Make a sacrifice. In some embodiments, the harboxydiene splicing regulator is linked to the cleavable portion of the linker by a self-sacrificing spacer unit. In certain embodiments, the harboxydiene splicing regulator is bound to the cleavable portion of the linker by a self-sacrificing spacer unit, the cleavable portion comprising Val-Cit and the maleimide caproyl (MC) cleavable portion. Tether to an antibody or antigen binding fragment. In certain embodiments, the harboxydiene splicing regulator is bound to the cleavable portion of the linker by a self-sacrificing spacer unit, the cleavable portion comprising Val-Ala and the maleimide caproyl (MC) cleavable portion. Tether to an antibody or antigen binding fragment. In certain embodiments, the harboxydiene splicing regulator is bound to the cleavable portion of the linker by a self-sacrificing spacer unit, which cleavable portion comprises Glu-Val-Cit and is cleavable to maleimide caproyl (MC). The moieties are tethered to an antibody or antigen binding fragment. In certain embodiments, the harboxydiene splicing regulator is an antibody or antibody via a Mal-spacer unit (eg, MC) in a linker tethered to a pABC or pAB self-sacrificing spacer unit and a Val-Cit cleavable moiety. It is coupled to an antigen-binding fragment. In certain other embodiments, the harboxydiene splicing regulator is via a pABC or pAB self-sacrificing spacer unit and a Mal-spacer unit (eg, MC) in a linker tethered to a Val-Ala cleavable moiety. It is coupled to an antibody or antigen binding fragment. In certain other embodiments, the harboxydiene splicing regulator comprises a pABC or pAB self-sacrificing spacer unit and a Mal-spacer unit (eg, MC) in a linker tethered to a Glu-Val-Cit cleavable moiety. It is conjugated to an antibody or antigen binding fragment via.

In certain embodiments, the self-sacrificing spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, p-aminobenzyl alcohol (pABOH) is linked to an amino acid unit or other cleavable moiety in the linker via an amide bond, and between pABOH and the drug moiety is a carbamate, methylcarbamic acid. Salts, or carbonates, are made (Hamann et al. (2005) Expert Opin Ther Patients 15: 1087-103). In some embodiments, the self-sacrificing spacer unit is or comprises p-aminobenzyloxycarbonyl (pABC). Although not constrained by theory, pABC self-sacrifice appears to involve a spontaneous 1,6-elimination reaction (Jin et al. (2015) Palm Res. 32: 3526-40).

In various embodiments, the structure of p-aminobenzyloxycarbonyl (pABC) used in the disclosed ADCs is shown below:

In various embodiments, the self-sacrificing spacer unit ties the cleavable portion of the linker to a harboxydiene splicing regulator. In some embodiments, the self-sacrificing spacer unit is pABC. In some embodiments, pABC ligates the cleavable portion of the linker to a harboxydiene splicing regulator. In some embodiments, the pABC is self-sacrificing upon cleavage of the cleavable portion and the harboxydiene splicing regulator is released from the ADC in its undenatured active form.

In some embodiments, the anti-HER2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Cit-pABC. In other embodiments, the anti-HER2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Ala-pABC.

In some embodiments, the anti-CD138 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Cit-pABC. In another embodiment, the anti-CD138 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Ala-pABC.

In some embodiments, the anti-EPHA2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Cit-pABC. In other embodiments, the anti-EPHA2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Ala-pABC.

In some embodiments, pABC self-sacrifices upon cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises the amino acid unit-pABC. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

In some embodiments, pABC self-sacrifices upon cleavage of the cleavable β-glucuronide moiety in the linker. In some embodiments, the linker comprises β-glucuronide-pABC.

In certain embodiments, the self-sacrificing spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, the self-sacrificing spacer unit in the linker comprises p-aminobenzyl (pAB). In some embodiments, the self-sacrifice of pAB involves a spontaneous 1,6-elimination reaction.

In various embodiments, the structure of p-aminobenzyl (pAB) used in the disclosed ADCs is shown below:

In various embodiments, the self-sacrificing spacer unit ties the cleavable portion of the linker to a harboxydiene splicing regulator. In some embodiments, the self-sacrificing spacer unit is pAB. In some embodiments, pAB binds the cleavable portion of the linker to a harboxydiene splicing regulator. In some embodiments, pAB is self-sacrificing upon cleavage of the cleavable portion and the harboxydiene splicing regulator is released from the ADC in its undenatured active form.

In some embodiments, the anti-HER2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Cit-pAB. In other embodiments, the anti-HER2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Ala-pAB.

In some embodiments, the anti-CD138 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Cit-pAB. In another embodiment, the anti-CD138 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Ala-pAB.

In some embodiments, the anti-EPHA2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Cit-pAB. In other embodiments, the anti-EPHA2 antibody or antigen binding fragment is coupled to the harboxidiene splicing regulator by a linker containing MC-Val-Ala-pAB.

In some embodiments, pAB self-sacrifices upon cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises the amino acid unit-pAB. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pAB. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pAB.

In some embodiments, pAB self-sacrifices upon cleavage of the cleavable β-glucuronide moiety in the linker. In some embodiments, the linker comprises β-glucuronide-pAB.

In some other embodiments, the harboxydiene splicing regulator is linked to the cleavable portion of the linker by a non-self-sacrificing spacer unit. In certain embodiments, the harboxydiene splicing regulator is bound to the cleavable portion of the linker by a non-self-sacrificing spacer unit, the cleavable portion comprising Val-Cit and the maleimide caproyl (MC) cleavable moiety. To the antibody or antigen-binding fragment. In certain embodiments, the harboxydiene splicing regulator is bound to the cleavable portion of the linker by a non-self-sacrificing spacer unit, the cleavable portion comprising Val-Ala and the maleimide caproyl (MC) cleavable moiety. To the antibody or antigen-binding fragment.

In various embodiments, the antibody or antigen binding fragment of the ADC is conjugated to the harboxydiene splicing regulatory drug moiety via a linker, where the linker is a Mal-spacer unit (eg, MC) and a cleavable amino acid unit. And pABC. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises maleimide caproyl (MC). In some embodiments, the linker comprises Mal-spacer unit-amino acid unit-pABC. In some embodiments, the linker comprises MC-amino acid unit-pABC. In some embodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC.

In various other embodiments, the antibody or antigen binding fragment of the ADC is conjugated to the harboxydiene splicing regulatory drug moiety via a linker, where the linker is cleavable with a Mal-spacer unit (eg, MC). Includes amino acid units and pAB. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises maleimide caproyl (MC). In some embodiments, the linker comprises Mal-spacer unit-amino acid unit-pAB. In some embodiments, the linker comprises MC-amino acid unit-pAB. In some embodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments, the linker comprises MC-Glu-Val-Cit-pAB. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pAB.

In various other embodiments, the antibody or antigen binding fragment of the ADC is conjugated to the harboxidiene splicing regulatory drug moiety via a linker, where the linker is cleavable with a Mal-spacer unit (eg, MC). Includes β-glucuronide and pABC. In some embodiments, the linker comprises the Mal-spacer unit-β-glucuronide-pABC. In some embodiments, the linker comprises MC-β-glucuronide-pABC.

In yet another embodiment, the antibody or antigen binding fragment of the ADC is conjugated to the harboxidiene splicing regulatory drug moiety via a linker, where the linker is cleavable β with a Mal-spacer unit (eg, MC). -Includes glucuronide and pAB. In some embodiments, the linker comprises the Mal-spacer unit-β-glucuronide-pAB. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

In various embodiments, the ADC compound is of formula (I) :.
Ab- (L-H) _p (I)
[In the formula, Ab is an antibody or antigen binding fragment that targets neoplastic cells;
H is a harboxydiene splicing regulator;
L is a linker that covalently binds Ab to D; and p is an integer of 1-15].

In some embodiments, the antibody or antigen binding fragment (Ab) of the ADC is conjugated to the harboxydiene splicing regulatory drug moiety via a linker, wherein the linker is disclosed herein or by reference. Includes one or more components of any of the linkers incorporated, or any of the linkers disclosed herein or incorporated by reference.

In some embodiments, the linker comprises a cleavable moiety that is positioned such that no portion of the linker or antibody or antigen binding fragment remains bound to the harboxydiene splicing regulator after cleavage. In some embodiments, the cleavable moiety is a cleavable peptide moiety, eg, an amino acid unit such as Val-Cit or Val-Ala. In some embodiments, the amino acid unit or linker comprises Val-Cit. In some embodiments, the amino acid unit or linker comprises Val-Ala. In some embodiments, the amino acid unit or linker comprises Glu-Val-Cit.

In some embodiments, the linker comprises at least one spacer unit that binds the antibody or antigen binding fragment to the cleavable moiety. In some embodiments, the linker comprises at least one spacer unit that binds the antibody or antigen binding fragment to the drug moiety. In some embodiments, the spacer unit or linker comprises at least one alkyl moiety.

In some embodiments, the spacer unit in the linker is linked to the antibody or antigen binding fragment via the Mal moiety (“Mal-spacer unit”). In some embodiments, the Mal-spacer unit comprises at least one alkyl moiety. In some embodiments, the linker comprises maleimide caproyl (MC). In some embodiments, the linker comprises Mal- (CH ₂ ) ₂ (“Mal-Et”). In some embodiments, the linker comprises Mal- (CH ₂ ) ₆ (“Mal-Hex”). In some embodiments, the linker comprises Mal- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ ("Mal-Et-O-Et"). In some embodiments, the linker comprises Mal- (PEG) ₂ -CO. In some embodiments, the Mal-spacer unit binds an antibody or antigen binding fragment to the drug moiety.

In some embodiments, the Mal-spacer unit or linker is Mal- (PEG) ₂ , Mal- (PEG) ₃ , Mal- (PEG) ₄ , Mal- (PEG) ₅ , Mal- (PEG) ₆ , Includes Mal- (PEG) ₇ or Mal- (PEG) ₈ . In some embodiments, the Mal-spacer unit or linker comprises Mal- (PEG) ₂ . In some embodiments, the Mal-spacer unit or linker is Mal- (PEG) ₂ -CO, Mal- (PEG) ₃ -CO, Mal- (PEG) ₄ -CO, Mal- (PEG) ₅ -CO. , Mal- (PEG) ₆ -CO, Mal- (PEG) ₇ -CO, or Mal- (PEG) ₈ -CO. In some embodiments, the Mal-spacer unit or linker comprises Mal- (PEG) ₂ -CO. In some embodiments, the Mal-spacer unit or linker comprises Mal- (PEG) ₂ -CO and at least one additional spacer unit. In some embodiments, Mal- (PEG) ₂ -CO binds an antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises or consists of Mal- (PEG) ₂ -CO. Examples of "Mal- (PEG) ₂ -CO" linkers are also referred to herein as "ADL2" or "ADL2" linkers.

In some embodiments, the Mal-spacer unit or linker comprises MC. In some embodiments, the Mal-spacer unit or linker comprises an MC and at least one additional spacer unit. In some embodiments, the MC binds an antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises or consists of MC. Examples of "MC" linkers are also referred to herein as "ADL10" or "ADL10" linkers.

In some embodiments, the Mal-spacer unit or linker comprises Mal- (CH ₂ ) ₆ (“Mal-Hex”). In some embodiments, the Mal-spacer unit or linker comprises Mal-Hex and at least one additional spacer unit. In some embodiments, Mal-Hex binds an antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Hex. Examples of "Mal-Hex" linkers are also referred to herein as "ADL12" or "ADL12" linkers.

In some embodiments, the Mal-spacer unit or linker comprises Mal- (CH ₂ ) ₂ (“Mal-Et”). In some embodiments, the Mal-spacer unit or linker comprises Mal-Et and at least one additional spacer unit. In some embodiments, Mal-Et binds an antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Et. Examples of "Mal-Et" linkers are also referred to herein as "ADL14" or "ADL14" linkers.

In some embodiments, the Mal-spacer unit or linker comprises Mal- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ (“Mal-Et-O-Et”). In some embodiments, the Mal-spacer unit or linker comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, Mal-Et-O-Et binds an antibody or antigen binding fragment to a drug moiety. In some embodiments, the linker comprises Mal-Et-O-Et. Examples of "Mal-Et-O-Et" linkers are also referred to herein as "ADL15" or "ADL15" linkers.

In some other embodiments, the Mal-spacer unit binds an antibody or antigen binding fragment to the cleavable portion of the linker. In some embodiments, the cleavable moiety of the linker is a cleavable peptide moiety, eg, an amino acid unit. In some embodiments, the cleavable peptide moiety is Val-Cit or Val-Ala. In some embodiments, the Mal-spacer unit or linker comprises MC. In some embodiments, the linker comprises MC-Val-Cit. In some embodiments, the linker comprises MC-Val-Ala. In some embodiments, the linker comprises MC-Glu-Val-Cit. In some embodiments, the linker comprises MC-Ala-Ala-Asn.

In some embodiments, the spacer unit binds the cleavable portion of the linker to a harboxydiene splicing regulator. In some embodiments, the spacer unit that binds the cleavable moiety to the harboxydiene splicing regulator is self-sacrificing.

In some embodiments, the spacer unit comprises pABC. In some embodiments, pABC associates the cleavable moiety with a harboxydiene splicing regulator. In some embodiments, the cleavable moiety is a cleavable peptide moiety, eg, an amino acid unit. In some embodiments, the linker comprises the amino acid unit-pABC.

In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the linker comprises Val-Cit-pABC and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments, the linker comprises MC-Val-Cit-pABC and at least one additional spacer unit. Examples of MC-Val-Cit-pABC linkers are also referred to herein as "ADL1" or "ADL1" linkers.

In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Val-Ala-pABC and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments, the linker comprises MC-Val-Ala-pABC and at least one additional spacer unit. Examples of MC-Val-Ala-pABC linkers are also referred to herein as "ADL6" or "ADL6" linkers.

In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC and at least one additional spacer unit. Examples of MC-Glu-Val-Cit-pABC linkers are also referred to herein as "ADL23" or "ADL23" linkers.

In some embodiments, the linker comprises Ala-Ala-Asn-pABC. In some embodiments, the linker comprises Ala-Ala-Asn-pABC and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC and at least one additional spacer unit. Examples of MC-Ala-Ala-Asn-pABC linkers are also referred to herein as "ADL21" or "ADL21" linkers.

In some other embodiments, the spacer unit comprises pAB. In some embodiments, pAB associates the cleavable moiety with a harboxydiene splicing regulator. In some embodiments, the cleavable moiety is a cleavable peptide moiety, eg, an amino acid unit. In some embodiments, the linker comprises the amino acid unit-pAB.

In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the linker comprises Val-Ala-pAB and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments, the linker comprises MC-Val-Ala-pAB and at least one additional spacer unit. Examples of MC-Val-Ala-pAB linkers are also referred to herein as "ADL5" or "ADL5" linkers.

In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments, the linker comprises MC-Val-Cit-pAB and at least one additional spacer unit. Examples of MC-Val-Cit-pAB linkers are also referred to herein as "ADL7" or "ADL7" linkers.

In some embodiments, the linker comprises β-glucuronide-pABC. In some embodiments, the linker comprises β-glucuronide-pABC and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pABC. In some embodiments, the linker comprises MC-β-glucuronide-pABC and at least one additional spacer unit. Examples of MC-β-glucuronide-pABC are also referred to herein as "ADL13" or "ADL13" linkers.

In some embodiments, the linker comprises β-glucuronide-pAB. In some embodiments, the linker comprises β-glucuronide-pAB and an MC Mal-spacer unit that binds the linker to an antibody or antigen binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

In some embodiments, the antibody or antigen binding fragment is conjugated to the harboxidiene splicing regulatory drug moiety via the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker. Be gated. In various embodiments, the ADCs disclosed herein include ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linkers and harboxydiene splicing regulatory drug moieties. , Have been found to demonstrate desirable properties for therapeutic ADCs. In various embodiments, their properties are, but are not limited to, effective drug loading levels, low aggregation levels, under storage conditions or in vivo when compared to ADCs with other linker-loadages. Stability when in circulation (eg, serum stability), retention of affinity for target-expressing cells comparable to non-conjugated antibodies, strong cytotoxicity to target-expressing cells, low off-target cell killing levels , High levels of bystander killing, and / or effective in vivo anti-cancer activity. For example, in various embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker and harboxydiene splicing regulatory drug moiety disclosed herein are included. ADCs exhibit an increased ability to inhibit the growth and / or proliferation of target-expressing cells when compared to ADCs with other linker-payloads (eg, ADL10 linker and herboxydiene splicing regulatory drug moiety). In various embodiments, the ADCs disclosed herein include ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linkers and harboxydiene splicing regulatory drug moieties. Surprisingly, raw when compared to other splicing regulator-based ADCs (eg, Tyranthatin A-based ADCs, eg, as reported in Puthenwetil et al. Bioconjugate Chem. (2016) 27: 1880-8). It exhibits increased internal stability (eg, plasma stability).

In some embodiments, specific ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linkers and harboxydiene splicing regulator drug moieties disclosed herein. Good or superior functional properties provided by the combination can be observed with a linker-payload conjugated to an anti-HER2 antibody such as trastuzumab; an anti-CD138 antibody such as BB4; or an anti-EPHA2 antibody such as 1C1. ..

In some embodiments, the ADC is an antibody or antigen binding comprising an ADL1-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL2-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL5-harboxidien splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL6-harboxidien splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL7-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL12-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL13-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL14-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments. In some embodiments, the ADC is an antibody or antigen binding comprising an ADL15-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that retains the ability to target and internalize neoplastic cells. Includes fragments.

In some embodiments, the ADC comprises an ADL1-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL2-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL5-harboxidien splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL6-harboxidien splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL7-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL12-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL13-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL14-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells. In some embodiments, the ADC comprises an ADL15-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells.

In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing HER2 is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplasm cell expressing HER2 is an amino acid sequence of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3). Three heavy chain complementarity determining regions (HCDRs) comprising; three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3). LCDR) and included.

In some embodiments, the ADC is the formula (I) :.
Ab- (L-H) _p (I)
[During the ceremony:
(I) Ab is three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3); SEQ ID NO: 4 (LCDR1). ), SEQ ID NO: 5 (LCDR2), and an anti-HER2 antibody or antigen-binding fragment thereof comprising three light chain complementarity determining regions (LCDRs) comprising the amino acid sequence of SEQ ID NO: 6 (LCDR3);
(Ii) H is a harboxydiene splicing regulator;
(Iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer of 1-15]. Have.

In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing HER2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20. Includes variable region. In some embodiments, an antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing HER2 comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is trastuzumab. In some embodiments, p is an integer of 1-10, 2-8, or 4-8. In some embodiments, p is 4. In some embodiments, p is 8.

In some embodiments, the ADC comprises an ADL1-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL2-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL5-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL6-harboxidien splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL7-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL12-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL13-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL14-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, the ADC comprises an ADL15-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138.

In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing CD138 is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing CD138 is the amino acid sequence of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3). Three heavy chain complementarity determining regions (HCDRs) comprising; three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3). LCDR) and included.

In some embodiments, the ADC is the formula (I) :.
Ab- (L-H) _p (I)
[During the ceremony:
(I) Ab is with three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); SEQ ID NO: 10 (LCDR1). ), An anti-CD138 antibody or antigen-binding fragment thereof comprising three light chain complementarity determining regions (LCDRs) comprising the amino acid sequence of SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3);
(Ii) H is a harboxydiene splicing regulator;
(Iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer of 1-15]. Have.

In some embodiments, the antibody or antigen binding fragment thereof that targets a neoplastic cell expressing CD138 is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 22. Includes variable region. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing CD138 comprises a mouse IgG2a heavy chain constant domain and a mouse Ig kappa light chain constant domain. In some embodiments, an antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing CD138 comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is BB4. In some embodiments, p is an integer of 1-10, 2-8, or 4-8. In some embodiments, p is 4. In some embodiments, p is 8.

In some embodiments, the ADC comprises an ADL1-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL2-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL5-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL6-harboxidien splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL7-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL12-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL13-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL14-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, the ADC comprises an ADL15-harboxydiene splicing regulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2.

In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing EPHA2 is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplasm cell expressing EPHA2 is an amino acid sequence of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3). Three heavy chain complementarity determining regions (HCDRs) comprising; three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3). LCDR) and included.

In some embodiments, the ADC is the formula (I) :.
Ab- (L-H) _p (I)
[During the ceremony:
(I) Ab is with three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); SEQ ID NO: 16 (LCDR1). ), An anti-EPHA2 antibody or antigen-binding fragment thereof comprising three light chain complementarity determining regions (LCDRs) comprising the amino acid sequence of SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3);
(Ii) H is a harboxydiene splicing regulator;
(Iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer of 1-15]. Have.

In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing EPHA2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 24. Includes variable region. In some embodiments, an antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing EPHA2 comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is 1C1. In some embodiments, p is an integer of 1-10, 2-8, or 4-8. In some embodiments, p is 4. In some embodiments, p is 8.

Harboxydiene Splicing Modulator In some embodiments, the antibody-drug conjugate is the formula (I): Ab- (L-H) _p [in the formula, Ab is an antibody or antigen that targets a neoplastic cell. Bound fragment; H is a harboxydiene splicing regulator; L is a linker that covalently binds Ab to H; and p is an integer of 1-15] antibody-drug conju. It is a gate.

又はその薬学的に許容可能な塩［式中：
Ｙは、Ｏ、Ｓ、ＮＲ^６、及びＣＲ^６Ｒ^７から選択され；
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^４は、水素、Ｃ_１～Ｃ_６アルキル基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、及び－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^５は、水素、ヒドロキシル、－ＣＨ_２－ＯＨ、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、－Ｏ－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、及び－ＮＲ^６－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｒ^５、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され、及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (I) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
Y is selected from O, S, NR ⁶ , and CR ⁶ R ⁷ ;
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁴ is hydrogen, C ₁ to C ₆ alkyl group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C. Selected from (= O)-(C ₃ to C ₈ heterocyclyl) groups and -C (= O) -NR ⁶ R ⁷ ;
R ⁵ is hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C (= O) -O- (C ₁ to C ₆ alkyl) group, -C (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , -OC (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , and -NR ⁶ -C (= O) -Selected from NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ and -C (= O) -OR ⁸ ; and R ⁸ is C ₁ Selected from ~ C ₆ alkyl groups, C ₃ ~ C ₈ carbocyclyl groups, and C ₃ ~ C ₈ heterocyclyl groups.
Here, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and —O— (C ₁ ). -C ₆ alkyl) group, -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ ). -C ₃ alkyl) and -NH-C (= O) -O- (may be independently substituted with 0 or 1 group selected from C ₁ to C ₃ alkyl)). It is substituted with 0 to 3 groups and does not exceed the valence of the atom covalently attached to L here].

又はその薬学的に許容可能な塩［式中：
Ｒ^９は、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され；
Ｒ^１０は、Ｈ及びＣ_１～Ｃ_６アルキル基から選択され、
ここでＲ^９及びＲ^１０は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、－ＮＨ_２、－ＮＨ－（Ｃ_１～Ｃ_３アルキル）、及び－Ｎ－（Ｃ_１～Ｃ_３アルキル）_２から独立に選択される０～３個の基で置換され、及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (Ia) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ⁹ is selected from C ₃ to C ₈ heterocyclyl groups;
R ¹⁰ is selected from H and C ₁ to C ₆ alkyl groups.
Here, R ⁹ and R ¹⁰ are independently halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, -NH ₂ , -NH- (C ₁ to C ₃ alkyl), and -N- (C ₁ to C ₃ alkyl) substituted with 0 to 3 groups independently selected from ₂ , and where the valence of the atom covalently attached to L is not exceeded] including.

又はその薬学的に許容可能な塩［式中：
Ｒ^１１は、

（式中、^＊は、Ｒ^１１が化合物の残りの部分に結合する点を表す）から選択され；
Ｒ^１２及びＲ^１３は、各々独立に、Ｈ及びメチルから選択され；及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (Ib) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ¹¹ is

(In the formula, ^* represents the point where R ¹¹ binds to the rest of the compound);
R ¹² and R ¹³ are each independently selected from H and methyl; and here do not exceed the valence of the atom covalently attached to L].

又はその薬学的に許容可能な塩［式中：
Ｘは、ヒドロキシル又はＮＲ^６Ｒ^７であり；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、－Ｃ（＝Ｏ）－Ｏ－Ｒ^８、－（Ｃ_１～Ｃ_６アルキル）－Ｏ－Ｃ（＝Ｏ）－Ｒ^８、及び－（Ｃ_１～Ｃ_６アルキル）－ＮＨ－Ｃ（＝Ｏ）－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され、及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (II) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
X is hydroxyl or NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently hydrogen, -R ⁸ , -C (= O) -R ⁸ , -C (= O) -OR ⁸ ,-(C ₁ to C ₆ alkyl) -O. -C (= O) -R ⁸ and-(C ₁ to C ₆ alkyl) -NH-C (= O) -R ⁸ ; and R ⁸ is a C ₁ to C ₆ alkyl group, C ₃ Selected from the ~ _{C8 carbocyclyl} group and the C3 ~ _C8 heterocyclyl group.
Here, R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, -O- (C ₁ to C ₆ alkyl) groups, -CO ₂ H, and -C (, respectively. = O) -O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl Groups and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O). ) (C1 to C3 alkyl) and -NH _{- C (} = O) -O- ₍ C1 to C3 alkyl) may be independently substituted with 0 or 1 group selected from them). It is substituted with 0 to 3 groups independently selected from, and does not exceed the valence of the atom covalently attached to L here].

又はその薬学的に許容可能な塩［式中：
Ｚは、ＮＲ^９及びＯから選択され；
Ｒ^９は、水素及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^１０及びＲ^１１は、各々独立に、水素、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；
Ｒ^１２は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^９、Ｒ^１０、Ｒ^１１、及びＲ^１２は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、及びＣ_１～Ｃ_３ハロアルキル基から選択される０又は１個の基で置換され；
ｔは、１、２、３、４、５、及び６から選択される整数であり；及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (IIa) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
Z is selected from NR ⁹ and O;
R ⁹ is selected from hydrogen and C ₁ to C ₆ alkyl groups;
R ¹⁰ and R ¹¹ are independently hydrogen, halogen, hydroxyl, C ₁ to C ₆ alkyl group, -O- (C ₁ to C ₆ alkyl) group, -CO ₂ H, -C (= O)-. O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl group. Selected from;
R ¹² is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups.
Here, R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, and C ₁ to C ₃ haloalkyl group, respectively. Substituted with 0 or 1 group to be;
t is an integer selected from 1, 2, 3, 4, 5, and 6; and here does not exceed the valence of the atom covalently attached to L].

又はその薬学的に許容可能な塩［式中：
Ｒ^１３は、

（式中、^＊は、Ｒ^１３が化合物の残りの部分に結合する点を表す）から選択され；
Ｒ^１４及びＲ^１５は、各々独立に、水素及びメチルから選択され；及び
ここでＬに共有結合的に結び付いている原子の原子価を超えることはない］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (IIb) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ¹³ is

( ^In the formula, ^* represents the point where R13 binds to the rest of the compound);
R ¹⁴ and R ¹⁵ are each independently selected from hydrogen and methyl; and here do not exceed the valence of the atom covalently attached to L].

又はその薬学的に許容可能な塩［式中：
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；及び
Ｒ^９は、Ｈ、

から選択され；
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され、
ここでＬに共有結合的に結び付いている原子の原子価を超えることはなく；及び
式中、^＊は、Ｒ^９が化合物の残りの部分に結合する点を表す］を含む。 In some embodiments, H, the harboxydiene splicing regulator, is a compound of formula (III) that is covalently linked to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ , and -C (= O) -OR ⁸ ;
R ⁸ is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups; and R ⁹ is H,

Selected from;
Here, R ¹ , R ² , R ³ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and -O- (C ₁ to C ₆ alkyl) groups, respectively. , -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ ) ~ C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ ~ C ₈ carbocyclyl group, C ₁ ~ C ₆ alkyl hydroxy group, C ₁ ~ C ₆ alkyl alkoxy group, benzyl group, and C ₃ ~ C ₈ heterocyclyl group. (Each of these is halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ to C ₃ alkyl), And 0 to 3 independently selected from 0 or 1 group selected from -NH-C (= O) -O- (C ₁ to C ₃ alkyl)). Replaced by the group of
Here it does not exceed the valence of the atom covalently attached to L; and in the equation, ^* represents the point where R ⁹ is attached to the rest of the compound].

In some embodiments, the antibody or antigen binding fragment is HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and / or STEP1. Target cells that express.

In some embodiments, the antibody or antigen binding fragment targets HER2-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-HER2 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1) comprising the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3). , HCDR2, and HCDR3); three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3). And include. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragment targets CD138 expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-CD138 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1) comprising the amino acid sequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3). , HCDR2, and HCDR3); three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3). And include. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a mouse Ig kappa light chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragment targets EPHA2-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-EPHA2 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1) comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3). , HCDR2, and HCDR3); three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3). And include. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragment targets MSLN expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-MSLN antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets FOLH1-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-FOLH1 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets CDH6-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-CDH6 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets CEACAM5-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-CEACAM5 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets CFC1B expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-CFC1B antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets ENPP3-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-ENPP3 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets FOLR1-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-FOLR1 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets HAVCR1-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-HAVCR1 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets KIT expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-KIT antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets MET-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-MET antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets MUC16 expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-MUC16 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets SLC39A6 expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-SLC39A6 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets SLC44A4 expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-SLC44A4 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets STEAP1-expressing cells. In some embodiments, the antibody or antigen binding fragment is an anti-STEAP1 antibody or antigen binding fragment.

In some embodiments, L is selected from any of the linkers disclosed herein, or any combination of linker components disclosed herein. In some embodiments, L is MC-Val-Cit-pABC, Mal- (PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB. , Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may also include one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker may also include one or more additional spacer units.

In certain embodiments, the intermediate, which is the precursor of the linker moiety, is reacted with the harboxidiene splicing regulator moiety under appropriate conditions. In certain embodiments, harboxydiene splicing regulators and / or reactive groups on intermediates or linkers are used. Subsequently, a reaction product between the harboxydiene splicing regulator and the intermediate, or a derivatized harboxydiene splicing regulator (harboxydiene splicing regulator + linker) was added to the antibody or antigen-binding fragment under appropriate conditions. React. Alternatively, the intermediate or linker may first react with the antibody or antigen-binding fragment, or the derivatized antibody or antigen-binding fragment, and then with the drug or derivatized drug.

A number of different reactions are available to covalently link the harboxydiene splicing regulatory and / or linker moieties to the antibody or antigen binding fragment. This is often one or more amino acid residues of an antibody or antigen binding fragment, including amine groups of lysine, free carboxylic acid groups of glutamate and aspartic acid, sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. Achieved by the reaction of the group. For example, a non-specifically covalently linked carbodiimide reaction linking a carboxy group (or amino group) on a harboxidien splicing regulator moiety to an amino group (or carboxy group) on an antibody or antigen binding fragment. It may be done using. In addition, bifunctional agents such as dialdehydes or imide esters may also be used to link amino groups on the harboxidiene splicing regulator moiety to amino groups on the antibody or antigen binding fragment. .. Schiff base reactions are also available to bind a drug (eg, a harboxydiene splicing regulator) to a binder. This method involves the oxidation of the periodic acid of a drug containing a glycol or hydroxy group, which in turn forms an aldehyde, which in turn reacts with the binder. Binding occurs by the formation of a Schiff base with the amino group of the binder. Isothiocyanate can also be used as a coupling agent to covalently bind the drug to the binder. Other techniques are known to those of skill in the art and are within the scope of this disclosure. Examples of drug moieties that can be made using a variety of chemistries known in the art and linked to antibodies or antigen binding fragments are harboxidiene splicing regulators, eg, described and exemplified herein. Harboxydiene splicing regulators.

Linker-Harboxidien Splicing Modulator / Harboxydien Splicing Modulator Compounds Further, the present specification includes exemplary Linker-Harboxidien Splicing Modulator (LH) compounds, as well as multiple copies of such compounds. The composition is disclosed. In various embodiments, the linker-harboxydiene splicing regulator compounds disclosed herein are according to the general formula: L-H [in the formula, L = linker moiety, and H = harboxydiene splicing regulator]. Can be defined. In certain embodiments, the disclosed LH compounds are suitable for use in the ADCs described herein.

又はその薬学的に許容可能な塩［式中：
Ｙは、Ｏ、Ｓ、ＮＲ^６、及びＣＲ^６Ｒ^７から選択され；
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^４は、水素、Ｃ_１～Ｃ_６アルキル基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、及び－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^５は、水素、ヒドロキシル、－ＣＨ_２－ＯＨ、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、－Ｏ－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７、－ＮＲ^６－Ｃ（＝Ｏ）－Ｒ^８、及び－ＮＲ^６－Ｃ（＝Ｏ）－ＮＲ^６Ｒ^７から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｒ^５、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換される］が開示される。 In some embodiments, the compound of formula (I) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
Y is selected from O, S, NR ⁶ , and CR ⁶ R ⁷ ;
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁴ is hydrogen, C ₁ to C ₆ alkyl group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C. Selected from (= O)-(C ₃ to C ₈ heterocyclyl) groups and -C (= O) -NR ⁶ R ⁷ ;
R ⁵ is hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C (= O) -O- (C ₁ to C ₆ alkyl) group, -C (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , -OC (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , and -NR ⁶ -C (= O) -Selected from NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ and -C (= O) -OR ⁸ ; and R ⁸ is C ₁ Selected from ~ C ₆ alkyl groups, C ₃ ~ C ₈ carbocyclyl groups, and C ₃ ~ C ₈ heterocyclyl groups.
Here, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and —O— (C ₁ ). -C ₆ alkyl) group, -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ ). -C ₃ alkyl) and -NH _- C ₍ = O) -O- (may be independently substituted with 0 or 1 group selected from C1 to C3 alkyl)). It is substituted with 0 to 3 groups to be substituted] is disclosed.

又はその薬学的に許容可能な塩［式中：
Ｒ^９は、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され；及び
Ｒ^１０は、Ｈ及びＣ_１～Ｃ_６アルキル基から選択され、
ここでＲ^９及びＲ^１０は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、－ＮＨ_２、－ＮＨ－（Ｃ_１～Ｃ_３アルキル）、及び－Ｎ－（Ｃ_１～Ｃ_３アルキル）_２から独立に選択される０～３個の基で置換される］が提供される。 In some embodiments, the present specification describes compounds of formula (Ia) that are covalently attached to L via any atom:

Or its pharmaceutically acceptable salt [in the formula:
R ⁹ is selected from the C ₃ to C ₈ heterocyclyl groups; and R ¹⁰ is selected from the H and C ₁ to C ₆ alkyl groups.
Here, R ⁹ and R ¹⁰ are independently halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, -NH ₂ , -NH- (C ₁ to C ₃ alkyl), and -N- (C ₁ to C ₃ alkyl) ₂ substituted with 0 to 3 groups independently selected] is provided.

又はその薬学的に許容可能な塩［式中：
Ｒ^１１は、

（式中、^＊は、Ｒ^１１が化合物の残りの部分に結合する点を表す）から選択され；
Ｒ^１２及びＲ^１３は、各々独立に、Ｈ及びメチルから選択される］が提供される。 In some embodiments, the compounds of formula (Ib) are described herein:

Or its pharmaceutically acceptable salt [in the formula:
R ¹¹ is

(In the formula, ^* represents the point where R ¹¹ binds to the rest of the compound);
R ¹² and R ¹³ are independently selected from H and Methyl, respectively].

又はその薬学的に許容可能な塩［式中：
Ｘは、ヒドロキシル又はＮＲ^６Ｒ^７であり；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、－Ｃ（＝Ｏ）－Ｏ－Ｒ^８、－（Ｃ_１～Ｃ_６アルキル）－Ｏ－Ｃ（＝Ｏ）－Ｒ^８、及び－（Ｃ_１～Ｃ_６アルキル）－ＮＨ－Ｃ（＝Ｏ）－Ｒ^８から選択され；及び
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換される］が提供される。 In some embodiments, the compound of formula (II) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
X is hydroxyl or NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently hydrogen, -R ⁸ , -C (= O) -R ⁸ , -C (= O) -OR ⁸ ,-(C ₁ to C ₆ alkyl) -O. -C (= O) -R ⁸ and-(C ₁ to C ₆ alkyl) -NH-C (= O) -R ⁸ ; and R ⁸ is a C ₁ to C ₆ alkyl group, C ₃ Selected from the ~ _{C8 carbocyclyl} group and the C3 ~ _C8 heterocyclyl group.
Here, R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, -O- (C ₁ to C ₆ alkyl) groups, -CO ₂ H, and -C (, respectively. = O) -O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl Groups and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O). ) (C1 to C3 alkyl) and -NH _{- C (} = O) -O- ₍ C1 to C3 alkyl) may be independently substituted with 0 or 1 group selected from them). Substituted with 0 to 3 groups independently selected from] is provided.

又はその薬学的に許容可能な塩［式中：
Ｚは、ＮＲ^９及びＯから選択され；
Ｒ^９は、水素及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^１０及びＲ^１１は、各々独立に、水素、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；
Ｒ^１２は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_３～Ｃ_８ヘテロシクリル基から選択され、
ここでＲ^９、Ｒ^１０、Ｒ^１１、及びＲ^１２は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、及びＣ_１～Ｃ_３ハロアルキル基から選択される０又は１個の基で置換され；及び
ｔは、１、２、３、４、５、及び６から選択される整数である］が提供される。 In some embodiments, the compounds of formula (IIa) are described herein:

Or its pharmaceutically acceptable salt [in the formula:
Z is selected from NR ⁹ and O;
R ⁹ is selected from hydrogen and C ₁ to C ₆ alkyl groups;
R ¹⁰ and R ¹¹ are independently hydrogen, halogen, hydroxyl, C ₁ to C ₆ alkyl group, -O- (C ₁ to C ₆ alkyl) group, -CO ₂ H, -C (= O)-. O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl group. Selected from;
R ¹² is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups.
Here, R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, and C ₁ to C ₃ haloalkyl group, respectively. Substituted with 0 or 1 group to be; and t is an integer selected from 1, 2, 3, 4, 5, and 6] is provided.

又はその薬学的に許容可能な塩［式中：
Ｒ^１３は、

（式中、^＊は、Ｒ^１３が化合物の残りの部分に結合する点を表す）から選択され；及び
Ｒ^１４及びＲ^１５は、各々独立に、水素及びメチルから選択される］が提供される。 In some embodiments, the compound of formula (IIb) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
R ¹³ is

(In the formula, ^* represents the point where R ¹³ binds to the rest of the compound); and R ¹⁴ and R ¹⁵ are independently selected from hydrogen and methyl, respectively]. ..

又はその薬学的に許容可能な塩［式中：
Ｒ^１、Ｒ^２、及びＲ^３は、各々独立に、水素、ヒドロキシル、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－Ｏ－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、及びＣ_１～Ｃ_６アルキル基から選択され；
Ｒ^６及びＲ^７は、各々独立に、水素、－Ｒ^８、－Ｃ（＝Ｏ）－Ｒ^８、及び－Ｃ（＝Ｏ）－Ｏ－Ｒ^８から選択され；
Ｒ^８は、Ｃ_１～Ｃ_６アルキル基、Ｃ_３～Ｃ_８カルボシクリル基、及びＣ_３～Ｃ_８ヘテロシクリル基から選択され；及び
Ｒ^９は、Ｈ、

から選択され、
ここでＲ^１、Ｒ^２、Ｒ^３、Ｒ^６、Ｒ^７、及びＲ^８は、各々独立に、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_６アルキル基、－Ｏ－（Ｃ_１～Ｃ_６アルキル）基、－ＣＯ_２Ｈ、－Ｃ（＝Ｏ）－（Ｃ_１～Ｃ_６アルキル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８カルボシクリル）基、－Ｃ（＝Ｏ）－（Ｃ_３～Ｃ_８ヘテロシクリル）基、－ＮＲ^６Ｒ^７、Ｃ_３～Ｃ_８カルボシクリル基、Ｃ_１～Ｃ_６アルキルヒドロキシ基、Ｃ_１～Ｃ_６アルキルアルコキシ基、ベンジル基、及びＣ_３～Ｃ_８ヘテロシクリル基（この各々は、ハロゲン、ヒドロキシル、Ｃ_１～Ｃ_３アルキル基、Ｃ_１～Ｃ_３アルコキシ基、Ｃ_１～Ｃ_３ハロアルキル基、－ＮＨ－Ｃ（＝Ｏ）（Ｃ_１～Ｃ_３アルキル）、及び－ＮＨ－Ｃ（＝Ｏ）－Ｏ－（Ｃ_１～Ｃ_３アルキル）から選択される０又は１個の基で独立に置換されていてもよい）から独立に選択される０～３個の基で置換され；及び
式中、^＊は、Ｒ^９が化合物の残りの部分に結合する点を表す］が提供される。 In some embodiments, the compound of formula (III) is described herein:

Or its pharmaceutically acceptable salt [in the formula:
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ , and -C (= O) -OR ⁸ ;
R ⁸ is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups; and R ⁹ is H,

Selected from
Here, R ¹ , R ² , R ³ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and -O- (C ₁ to C ₆ alkyl) groups, respectively. , -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ ) ~ C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ ~ C ₈ carbocyclyl group, C ₁ ~ C ₆ alkyl hydroxy group, C ₁ ~ C ₆ alkyl alkoxy group, benzyl group, and C ₃ ~ C ₈ heterocyclyl group. (Each of these is halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ to C ₃ alkyl), And 0 to 3 independently selected from 0 or 1 group selected from -NH-C (= O) -O- (C ₁ to C ₃ alkyl)). And in the formula, ^* represents the point where ^R9 binds to the rest of the compound] is provided.

In some embodiments, the present specification provides compound H1 or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H4, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H5, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H6, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H7, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H8, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H9, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H10, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H2, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H3, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H12, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H13, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H14, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H15, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H16, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H17, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H18, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H19, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H20, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H21, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H22, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H23, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H24, or a pharmaceutically acceptable salt thereof. In some embodiments, the present specification provides compound H25, or a pharmaceutically acceptable salt thereof.

及びその薬学的に許容可能な塩
（式中、Ｌは、抗体に共有結合的に結び付いているリンカーである）
から選択される化合物が提供される。 In some embodiments, the specification will include.

And its pharmaceutically acceptable salt (in the formula, L is a linker covalently attached to the antibody).
Compounds selected from are provided.

In some embodiments, the linker comprises at least one cleavable peptide moiety. In some embodiments, at least one cleavable peptide moiety is enzymatically cleavable. In some embodiments, the linker or cleavable peptide moiety comprises at least one amino acid unit. In some embodiments, the at least one amino acid unit is arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, aspartic acid, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, It is selected from phenylalanine, tyrosine, tryptophan, and citrulin. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the linker comprises citrulline and valine. In some embodiments, the linker comprises alanine and valine.

In some embodiments, the linker comprises a moiety selected from sulfonamides, β-glucuronides, disulfides, and carbonyls. In some embodiments, the linker comprises a sulfonamide. In some embodiments, the linker comprises β-glucuronide. In some embodiments, the linker comprises a disulfide. In some embodiments, the linker comprises a carbonyl.

In some embodiments, the linker comprises a spacer unit. In some embodiments, the spacer unit is selected from an alkyl group and a polyethylene glycol (PEG) moiety. In some embodiments, the alkyl group is a C ₁ to C ₁₂ alkyl group. In some embodiments, the alkyl group is a C ₁ to C ₆ alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG moiety comprises − (PEG) _m −, where m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

In some embodiments, the linker comprises a maleimide (Mal) moiety (“Mal-spacer unit”). In some embodiments, the linker comprises a self-sacrificing spacer unit. In some embodiments, the self-sacrificing spacer unit is selected from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

In some embodiments, the linker comprises a Mal-spacer unit, an alkyl group, at least one amino acid unit and a self-sacrificing spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit comprises alanine and valine. In some embodiments, at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-sacrificing spacer is selected from pAB and pABC. In some embodiments, the self-sacrificing spacer comprises pAB. In some embodiments, the self-sacrificing spacer comprises pABC. In some embodiments, the alkyl group comprises a C ₁ to C ₆ alkyl group.

In some embodiments, the linker comprises a Mal-spacer unit and a PEG moiety, at least one amino acid unit and a self-sacrificing spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit comprises alanine and valine. In some embodiments, at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-sacrificing spacer is selected from pAB and pABC. In some embodiments, the self-sacrificing spacer comprises pAB. In some embodiments, the self-sacrificing spacer comprises pABC. In some embodiments, the PEG moiety comprises − (PEG) _m −, where m is an integer from 1 to 6.

Drug loading The drug loading is represented by p and is also referred to herein as the harboxydiene splicing regulator to antibody ratio (HAR). The drug loading may range from 1 to 10 drug moieties per antibody or antigen binding fragment. In some embodiments, p is an integer of 1-10. In some embodiments, p is an integer of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. .. In some embodiments, p is an integer of 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3. In some embodiments, p is an integer of 1-8. In some embodiments, p is an integer of 2-5. In some embodiments, p is an integer of 2-4. In some embodiments, p is an integer of 3-4. In other embodiments, p is an integer of 4-8. In other embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 4 or 8.

Drug loading can be limited by the number of binding sites on the antibody or antigen binding fragment. In some embodiments, the linker moiety (L) of the ADC binds to the antibody or antigen binding fragment through a chemically active group on one or more amino acid residues on the antibody or antigen binding fragment. For example, the linker may be attached to an antibody or antigen-binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (eg, to the ε-amino group of one or more lysine residues at the N-terminus or C-terminus). It may be associated (to the free carboxylic acid group of one or more glutamate or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues). The site to which the linker is attached may be a natural residue in the amino acid sequence of the antibody or antigen binding fragment, or it may be, for example, by DNA recombination techniques (eg, by introducing a cysteine residue into the amino acid sequence). Alternatively, it may be introduced into an antibody or antigen-binding fragment by protein biochemistry (eg, by reduction, pH adjustment, or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugated to an antibody or antigen binding fragment is limited by the number of free cysteine residues. For example, if the binding is a cysteine thiol group, the antibody may have only one or a few cysteine thiol groups, or only one or a few sufficiently reactive thiol groups that can bind a linker. It may not have. In general, antibodies are low in free and reactive cysteine thiol groups that can be linked to the drug moiety. In fact, most cysteine thiol residues in antibodies are involved in either interchain or intrachain disulfide bonds. Thus, conjugation to cysteine may, in some embodiments, require at least partial reduction of the antibody. Overbinding of the linker-toxin to the antibody can destabilize the antibody by reducing the cysteine residues available for the formation of disulfide bonds. Therefore, the optimal drug: antibody ratio should increase the efficacy of the ADC (by increasing the number of drug moieties bound per antibody) without destabilizing the antibody or antigen binding fragment. In some embodiments, the optimum ratio can be 2, 4, 6, or 8.

In some embodiments, the antibody or antigen binding fragment is exposed to reducing conditions prior to conjugation in order to give rise to one or more free cysteine residues. In some embodiments, the antibody may be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP), thereby reacting. Sexual cysteine thiol groups can occur. An unpaired cysteine may be produced through partial reduction with a restricted molar equivalent of TCEP, which involves light chain and heavy chain (each HL pair formation) while leaving the intrachain disulfide bonds intact. It is possible to reduce interchain disulfide bonds that connect two heavy chains (two pairs for each HH pair formation in the case of human IgG1) and two heavy chains in the hinge region (Stefano et al. (One pair per) and two heavy chains in the hinge region. 2013) Methods Mol Biol. 1045: 145-71). In embodiments, the disulfide bonds in the antibody are electrochemically reduced, for example, by utilizing a working electrode that alternately applies reduction and oxidation voltages. This technique is an analytical device (eg, an electrochemical detector, an NMR spectrometer, or a mass spectrometer) or a chemical separation device (eg, liquid chromatography (eg, HPLC)) or an electrophoresis device (eg, US patent). (See Publication No. 20140069822))) may allow online binding of disulfide bond reduction. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophiles on amino acid residues, such as cysteine.

The drug loading of the ADC is otherwise, for example, (i) limiting the molar excess of the drug-linker intermediate or linker reagent to the antibody; (ii) limiting the conjugation reaction time or temperature; (iiii). ) Partial or restrictive reducing conditions for cysteine thiol modification; and / or (iv) antibodies such that the number and position of cysteine residues are modified to control the number and / or position of linker-drug associations. It may be controlled by modifying the amino acid sequence of.

In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody or antigen binding fragment. For example, a cysteine-modified antibody can be prepared in which one or more amino acids of the parent antibody are replaced with cysteine amino acids. Any form of antibody may be modified in this way, i.e. mutated. For example, the parent Fab antibody fragment may be modified to form a cysteine-modified Fab called "ThioFab". Similarly, the parent monoclonal antibody may be modified to form "ThioMab". In TioFab, a single site mutation yields a single modified cysteine residue, while in TioMab, due to the dimer nature of the IgG antibody, a single site mutation results in two modified cysteine residues. Give rise to a group. The DNA encoding the amino acid sequence variant of the parent polypeptide can be prepared by a variety of methods known in the art (see, eg, the method described in WO 2006/0344488). .. These methods include, but are not limited to, site-specific (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of the previously prepared DNA encoding the polypeptide. Induced preparation can be mentioned. Recombinant antibody variants may also be constructed by restriction enzyme fragment manipulation or by overlapping extension PCR with synthetic oligonucleotides. ADCs of formula (I) include, but are not limited to, antibodies having 1, 2, 3, or 4 modified cysteine amino acids (Lyon et al. (2012) Methods Enzymol. 502: 123-38). In some embodiments, the antibody or antigen binding fragment already has one or more free cysteine residues without modification, in which case the existing free cysteine residues are used to bind the antibody or antigen. Fragments can be conjugated to drug moieties.

If two or more nucleophilic groups react with a drug-linker intermediate or linker partial reagent followed by a drug partial reagent in a reaction mixture containing multiple copies of the antibody or antigen binding fragment and linker moiety, they are obtained there. The product can be a mixture of ADC compounds having a distribution of one or more drug moieties associated with each copy of an antibody or antigen binding fragment in the mixture. In some embodiments, the drug loading of the ADC mixture obtained from the conjugation reaction ranges from 1 to 10 drug moieties associated with each antibody or antigen binding fragment. The average number of drug moieties (ie, average drug loading, or average p) per antibody or antigen binding fragment is determined by any conventional method known in the art, such as mass spectrometry (eg, reverse phase LC-MS). And / or can be calculated by high performance liquid chromatography (eg, HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is determined by reverse phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is from about 1.5 to about 3.5, from about 2.5 to about 4.5, from about 3.5 to about 5.5. , About 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8. , About 7 to about 9, about 2 to about 8, or about 4 to about 8.

In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 2. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2. 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is 2.

In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about. 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is 4.

In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 8. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about. 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is 8.

In various embodiments, the term "about" means ± 10% when used with respect to the average number of drug moieties per antibody or antigen binding fragment.

Individual ADC compounds, or "species", can be identified in the mixture by mass spectral method and separated by UPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC). In certain embodiments, for example by electrophoresis or chromatography, a single loading value uniform or near uniform ADC product can be separated from the conjugation mixture.

In some embodiments, high drug loading (eg, p> 8) can result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. Higher drug loadings can also adversely affect the pharmacokinetics (eg, clearance) of certain ADCs. In some embodiments, lower drug loadings (eg, p <2) can reduce the efficacy of certain ADCs on target expressing cells and / or bystander cells. In some embodiments, the drug loadings of the ADCs of the present disclosure are about 2 to about 8; about 2 to about 6; about 2 to about 5; about 3 to about 5; about 2 to about 4; or about 4. It is in the range of about 8.

In some embodiments, for example, partial reduction of intrachain disulfides on an antibody or antigen binding fragment is used to achieve about 2 drug loadings and / or average drug loadings, which provides beneficial properties. In some embodiments, for example, partial reduction of intrachain disulfides on an antibody or antigen binding fragment is used to achieve about 4 drug loadings and / or average drug loadings, which provides beneficial properties. In some embodiments, for example, partial reduction of intrachain disulfides on an antibody or antigen binding fragment is used to achieve about 8 drug loadings and / or average drug loadings, which provides beneficial properties. In some embodiments, deconjugation where the drug loading and / or average drug loading is less than about 2 can compete with the ADC for binding to the target antigen and / or result in diminished therapeutic efficacy. The level of antibody species can be unacceptably high. In some embodiments, when the drug loading and / or average drug loading exceeds about 8, the level of product heterogeneity and / or ADC aggregation can be unacceptably high. When the drug loading and / or average drug loading exceeds about 8, the stability of the ADC is also due to the loss of one or more chemical bonds required to stabilize the antibody or antigen binding fragment. Can also be affected.

The present disclosure includes the described method of making an ADC. Briefly, the ADC is an antibody or antigen-binding fragment such as the antibody or antigen-binding fragment, a drug moiety (eg, a harboxydiene splicing regulator), and a linker that binds the drug moiety to the antibody or antigen-binding fragment. including. In some embodiments, the ADC can be prepared with a linker having a reactive functional group for covalent binding to the drug moiety and antibody or antigen binding fragment. For example, in some embodiments, the cysteine thiol of the antibody or antigen binding fragment can form a bond with the reactive functional group (eg, the maleimide moiety) of the linker or drug-linker intermediate to create an ADC. The creation of the ADC can be accomplished by any technique known to those of skill in the art.

In some embodiments, the ADC is made by sequentially contacting an antibody or antigen binding fragment with a linker and a drug moiety (eg, a harboxydiene splicing regulator), i.e., initially the antibody or antigen binding fragment is attached to the linker. It is covalently linked and then this preformed antibody-linker intermediate reacts with the drug moiety. The antibody-linker intermediate may or may not be subjected to the purification step prior to contact with the drug moiety. In another embodiment, the ADC is made by contacting a preformed linker-drug compound with an antibody or antigen binding fragment by reacting the linker with the drug moiety. The preformed linker-drug compound may or may not be subjected to a purification step prior to contact with the antibody or antigen binding fragment. In other embodiments, the antibody or antigen binding fragment is contacted with the linker and drug moiety in one reaction mixture to be covalent between the antibody or antigen binding fragment and the linker, and between the linker and the drug moiety. Simultaneous formation of bonds is achieved. This ADC preparation method may include contacting the antibody or antigen binding fragment with the antibody or antigen binding fragment prior to adding the linker to the reaction mixture, and vice versa. In certain embodiments, the ADC is an ADL1-harboxydiene splicing regulator (eg, ADL1-79392) or an ADL5-harboxydiene splicing regulator (eg, ADL5-0349), and the like, under conditions that allow conjugation. , Made by reacting an antibody or antigen binding fragment with a linker tethered to a drug moiety.

The ADC prepared as described above may be subjected to a purification step. The purification step may include any biochemical method known in the art for purifying proteins, or any combination of those methods. Such methods include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, chromatography based on any charge or isoelectric point, mixed mode chromatography, eg, CHT (ceramic hydroxy). Apatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

Therapeutic Uses and Compositions The present specification discloses methods of using the disclosed ADCs and compositions in the treatment of a subject for a disorder, eg, a neoplastic disorder. The ADC may be administered alone or in combination with a second therapeutic agent, or with any pharmaceutically acceptable formulation, dosage, and administration regimen. ADC therapeutic efficacy is assessed for toxicity as well as efficacy indicators and may be adjusted accordingly. Efficacy measures include, but are not limited to, cell proliferation inhibitory and / or cytotoxic effects observed in vitro or in vivo, tumor volume reduction, tumor proliferation inhibition rates, and / or prolongation of survival.

Methods for determining whether ADC has a cell proliferation inhibitory effect and / or a cytotoxic effect on cells are known. For example, the cytotoxic or inhibitory activity of ADCs is the exposure of mammalian cells expressing the target protein to ADCs in cell culture medium; culturing cells for a period of about 6 hours to about 6 days; and It can be measured by measuring cell viability. Cell-based in vitro assays may also be used to measure viability (proliferation), cytotoxicity, and ADC apoptosis induction (caspase activation).

A thymidine uptake assay may be used to determine if the ADC exerts a cell proliferation inhibitory effect. For example, cancer cells expressing a target antigen plated at a density of 5,000 cells per well in 96 wells were cultured for a period of 72 hours and 0.5 μCi for the last 8 hours of the 72 hour period. Can be exposed to ³ H-thymidine. Uptake of ^3H -thymidine into cells of culture is measured in the presence and absence of ADC.

Necrosis or apoptosis (programmed cell death) may be measured to determine cytotoxicity. Necrosis is typically associated with increased permeability of the cell membrane; swelling of the cell and rupture of the cell membrane. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercially available photometric methods are available that quantitatively determine DNA fragmentation in vitro. Examples of such assays include TUNEL, which detects the uptake of labeled nucleotides in fragmented DNA, and ELISA-based assays, Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis may also be determined by measuring morphological changes in cells. For example, as with necrosis, the loss of cell membrane integrity can be determined by measuring the uptake of certain dyes (eg, fluorescent dyes such as acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells is described by Duke and Cohen, Current Protocols in Immunology (Polypropylene et al., Eds. (1992) pp.3.17.1-3.17.16). The cells can also be labeled with a DNA dye (eg, acridine orange, ethidium bromide, or propidium iodide) and the cells can be observed for chromatin condensation and marginal orientation along the intima of the nucleus. Apoptosis may also be determined by screening for caspase activity in some embodiments. In some embodiments, the Caspase-Glo® assay can be used to measure the activity of caspase-3 and caspase-7. In some embodiments, the assay provides a luminescent caspase-3 / 7 substrate in reagents optimized for caspase activity, luciferase activity, and cytolysis. In some embodiments, the addition of Caspase-Glo® 3/7 reagent in the "add-mix-measure" format results in cytolysis, followed by caspase cleavage of the substrate, and luciferase. The generation of "glow-type" emission signals caused by the can occur. In some embodiments, luminescence may be proportional to the abundance of caspase activity and may serve as an indicator of apoptosis. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic aggregation, increased membrane bleb formation, and cell atrophy. Determination of any of these effects on cancer cells indicates that ADCs are useful in the treatment of cancer.

Cell viability may be measured, for example, by determining the uptake of a dye such as neutral red, trypan blue, crystal violet, or ALAMAR ™ blue in cells (eg, Page et al. (1993)). See Intl J Oncology 3: 473-6). In such an assay, the cells are incubated in a medium containing the dye, the cells are washed, and the residual dye that reflects the uptake of the dye by the cells is measured spectrophotometrically. Cell viability may also be measured, for example, by quantifying ATP, which is an indicator of cells with metabolic activity. In certain embodiments, the in vitro efficacy and / or cell viability of the prepared ADC or harboxydiene splicing regulator compound is CellTiter-Glo®, as described in the examples provided herein. It may be determined using a luminescent cell viability assay. In this assay, in certain embodiments, a single reagent (CellTiter-Glo® reagent) is added directly to cells cultured in serum supplement medium. Addition of reagents results in cytolysis and generation of luminescent signals proportional to the abundance of ATP. The amount of ATP is directly proportional to the number of cells present in culture. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst. 82: 1107-12).

The disclosed ADCs may also be evaluated for bystander killing activity. Bystander killing activity may be determined, for example, by an assay using two cell lines, one positive for the target antigen and one negative for the target antigen. In certain embodiments, the assay design allows tracking of only target-negative cells. In certain embodiments, cells are plated under three conditions: (i) target-negative cells alone (tagged or labeled); (ii) target-positive cells alone; and (iii) with target-negative cells. Co-culture with target positive cells. The cells are then treated with ADC and subsequently monitored for cytotoxicity. Reading the plate with the CellTiter-Glo® reagent allows monitoring the viability of all cell populations. When the plate is read with the OneGlo® reagent, only tagged or labeled target-negative cells generate a signal. Killing of target-negative cells when mixed with target-positive cells is an indicator of bystander killing, while killing of target-negative cells in the absence of target-positive cells is an indicator of off-target killing.

In certain embodiments, the present disclosure features methods of killing cancer cells or tissues by disrupting RNA splicing, inhibiting or regulating their growth, or interfering with their metabolism. The method can be used in any subject that benefits from treatment by disruption of RNA splicing. Targets where a disruption of RNA splicing can be beneficial include, but are not limited to, those who have or are at risk of having neoplastic disorders such as hematological malignancies or solid tumors. In certain embodiments, the hematological malignancies are B-cell malignancies, blood cancers (leukemias), plasma cell cancers (myeloma, eg multiple myeloma), or lymph node cancers (lymphomas). .. In certain embodiments, the hematological malignancies are acute myeloid leukemia or multiple myeloma. In certain embodiments, the leukemia is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelocytic leukemia (CLL), chronic myelocytic leukemia (CML), chronic myelomonocytic leukemia. Leukemia (CMML) or acute monocytic leukemia (AMOL). In certain embodiments, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. In certain embodiments, the hematological malignancies are myelodysplastic syndrome (MDS). In certain embodiments, solid tumors include breast cancer, pancreatic cancer, prostate cancer, colon or rectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, glioma, or melanoma. Cancer. In certain embodiments, solid tumors include breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous endometrial cancer), salivary tract cancer, melanoma, colon. Cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, or esophageal cancer. In certain embodiments, lung cancer is lung adenocarcinoma. In certain embodiments, the uterine cancer is serous endometrial cancer.

In various embodiments, the disclosed ADC may be administered in any cell or tissue expressing HER2, such as a HER2-expressing neoplastic cell or tissue. Exemplary embodiments include methods of inhibiting HER2-mediated cell signaling or killing cells. The method can be used on any cell or tissue that expresses HER2, such as cancerous cells or metastatic lesions. Non-limiting examples of HER2-expressing cancers include breast cancer, gastric cancer, bladder cancer, urinary epithelial cell cancer, esophageal cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous endometrial cancer). ), Salivary tract cancer, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diamond Ther. 17: 85-99). Non-limiting examples of HER2-expressing cells include HCC1954 and SKBR3 human ductal carcinoma cells, N87 human gastric cancer cells, and cells containing recombinant nucleic acids encoding HER2 or a portion thereof.

In various embodiments, the disclosed ADC may be administered in any cell or tissue expressing CD138, such as a CD138-expressing neoplastic cell or tissue. Exemplary embodiments include methods of inhibiting CD138-mediated cell signaling or killing cells. The method can be used on any cell or tissue that expresses CD138, such as cancerous cells or metastatic lesions. Non-limiting examples of cancers expressing CD138 include intrathoracic cancer (eg, lung cancer, mesotheloma), skin cancer (eg, basal cell cancer, squamous epithelial cancer), head and neck cancer (eg, laryngeal, lower). Physopharyngeal, nasopharyngeal), breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urinary epithelial cancer), hematological malignancies (eg, multiple myeloma) Such as myeloma, Hodgkin's lymphoma), and thyroid cancer (Szatmari et al. (2015) Dis Markers 2015: 796052). Non-limiting examples of CD138-expressing cells include MOLP8 human multiple myeloma cells and cells containing recombinant nucleic acid encoding CD138 or a portion thereof.

In various embodiments, the disclosed ADC may be administered in any cell or tissue expressing EPHA2, such as EPHA2-expressing neoplastic cells or tissues. Exemplary embodiments include methods of inhibiting EPHA2-mediated cell signaling or killing cells. The method can be used on any cell or tissue that expresses EPHA2, such as cancerous cells or metastatic lesions. Non-limiting examples of cancers expressing EPHA2 include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al). (2011) Expert Opin The Targets 15 (1): 31-51. Non-limiting examples of EPHA2-expressing cells include PC3 human prostate cancer cells and cells containing recombinant nucleic acids encoding EPHA2 or a portion thereof. Can be mentioned.

An exemplary method comprises contacting a cell with an effective amount, i.e., an ADC as described herein in an amount sufficient to kill the cell. The method can be used, for example, in vitro, in vivo, ex vivo, or in situ for cells in culture. For example, cells expressing HER2 (eg, cells recovered by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells) can be cultured in vitro in culture medium and contacted. The step of making can be affected by adding ADC to the culture medium. The method will result in the killing of HER2-expressing cells, including tumor cells expressing HER2 in particular. Alternatively, the ADC can be administered to the subject by any suitable route of administration (eg, intravenous, subcutaneous, or direct contact with tumor tissue) to exert its effect in vivo. This technique can be used for antibodies that target other cell surface antigens (eg, CD138, EPHA2).

The in vivo effects of the disclosed ADC therapeutic compositions can be assessed in a suitable animal model. For example, a heterologous cancer model may be used, where cancer explants or passaged xenograft tissues are introduced into immunodeficient animals such as nude mice or SCID mice (Klein et al. (1997) Nature Med. .3: 402-8). Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In vivo asses that assess the promotion of tumor death by mechanisms such as apoptosis can also be used. In one embodiment, xenografts from tumor-bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic focus and compared to untreated control xenograft-bearing mice. The extent to which apoptotic focus is found in treated mouse tumors provides an indicator of the therapeutic efficacy of the composition.

Further provided herein are methods of treating neoplastic disorders, such as cancer. The ADC disclosed herein can be administered to a non-human mammal or human subject for therapeutic purposes. Therapeutic methods are for targeting antibodies that bind to, are available for, or localize to, antigens expressed on the surface of cancer cells in subjects with or suspected of having neoplastic disorders. Accompanied by administering a therapeutically effective amount of an ADC or composition comprising a ligated herboxydiene splicing regulator. In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic cells adjacent to neoplastic cells that do not express the target antigen but express the target antigen.

An exemplary embodiment is a method of delivering a harboxidiene splicing regulator to cells expressing HER2, wherein the harboxidien splicing regulator is conjugated to an antibody that immunospecifically binds to a HER2 epitope. A method comprising exposing cells to ADC. Exemplary tumor cells expressing HER2 to which the ADC of the present disclosure is indicated include gastric cancer cells and ductal carcinoma cells.

Another exemplary embodiment is a method of delivering a harboxydiene splicing regulator to cells expressing CD138, in which the harboxydiensplicing regulator is conjugated to an antibody that immunospecifically binds to the CD138 epitope. , And methods comprising exposing cells to ADC. Exemplary tumor cells expressing CD138 to which the ADC of the present disclosure is indicated include multiple myeloma cells.

Another exemplary embodiment is a method of delivering a harboxydiene splicing regulator to cells expressing EPHA2, wherein the harboxydiensplicing regulator is conjugated to an antibody that immunospecifically binds to an EPHA2 epitope. , And methods comprising exposing cells to ADC. Exemplary tumor cells expressing EPHA2 to which the ADC of the present disclosure is indicated include prostate cancer cells.

Another exemplary embodiment is a method of reducing or inhibiting the growth of a tumor (eg, a HER2-expressing tumor, a CD138-expressing tumor, an EPHA2-expressing tumor), wherein a composition comprising a therapeutically effective amount of the ADC or ADC is administered. It is a method that includes that. In some embodiments, treatment reduces or inhibits tumor growth in a patient, reduces the number or size of metastatic lesions, reduces tumor load, reduces primary tumor load, and reduces invasiveness. Sufficient to prolong survival and / or maintain or improve quality of life. In some embodiments, the tumor is resistant or refractory to treatment with an antibody or antigen-binding fragment of ADC (eg, anti-HER2 antibody, anti-CD138 antibody, anti-EPHA2 antibody) when administered alone, and / or. Tumors are refractory or refractory to treatment with the drug portion of the herboxidien splicing regulator when administered alone.

In certain embodiments, the present disclosure provides methods of reducing or inhibiting the growth of HER2-expressing tumors. In certain embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of tumor cells adjacent to neoplastic tumor cells that do not express HER2 but do express HER2. Exemplary HER2-expressing tumor types are, but are not limited to, HER2-expressing breast cancer, gastric cancer, bladder cancer, urinary epithelial cell cancer, esophageal cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous). Endometrial cancer), salivary duct cancer, cervical cancer, endometrial cancer, and tumors derived from ovarian cancer. In certain embodiments, the HER2-expressing tumor is a HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous endometrial cancer), osteosarcoma, or salivary duct. It is a tumor derived from cancer. In certain embodiments, the HER2-expressing tumor is lung adenocarcinoma or serous endometrial cancer.

In certain embodiments, the present disclosure provides methods of reducing or inhibiting the growth of CD138-expressing tumors. In certain embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of tumor cells flanking the neoplastic tumor cells that do not express CD138 but do express CD138. Exemplary CD138-expressing tumor types are, but are not limited to, intrathoracic cancer expressing CD138 (eg, lung cancer, mesotheloma), skin cancer (eg, basal cell cancer, squamous epithelial cancer), head and neck cancer (eg, eg). , Laryngeal, hypopharyngeal, nasopharyngeal), breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urinary tract epithelial cancer), and tumors derived from thyroid cancer Can be mentioned.

In certain embodiments, the present disclosure provides methods of reducing or inhibiting the growth of EPHA2-expressing tumors. In certain embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of tumor cells flanking the neoplastic tumor cells that do not express EPHA2 but do express EPHA2. Exemplary EPHA2-expressing tumor types are derived from, but not limited to, EPHA2-expressing breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer. Examples include tumors. In certain embodiments, the EPHA2-expressing tumor is a tumor derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that expresses EPHA2.

In addition, the antibodies of the present disclosure may be administered to non-human mammals expressing antigens capable of binding ADCs for veterinary purposes or as an animal model of human disease. With respect to the latter, such animal models may be useful in assessing the therapeutic efficacy of the disclosed ADCs (eg, testing the dosage and time course of administration).

In addition, the present specification provides therapeutic uses of the disclosed ADCs and compositions. An exemplary embodiment is the use of ADC in the treatment of neoplastic disorders (eg, HER2-expressing cancer, CD138-expressing cancer, EPHA2-expressing cancer). Another exemplary embodiment is an ADC for use in the treatment of neoplastic disorders (eg, HER2-expressing cancer, CD138-expressing cancer, EPHA2-expressing cancer). Identification of a subject having a cancer expressing a target antigen (eg, HER2, CD138, EPHA2, MSLN, FULLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1). Is known in the art and can be used to identify patients suitable for treatment with the disclosed ADCs.

Another exemplary embodiment is the use of ADCs in methods of making therapeutic pharmaceuticals for neoplastic disorders (eg, HER2-expressing cancers, CD138-expressing cancers, EPHA2-expressing cancers).

The therapeutic composition used in carrying out the aforementioned method may be formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier suitable for the desired delivery method. An exemplary embodiment is a pharmaceutical composition comprising the ADCs of the present disclosure and a pharmaceutically acceptable carrier. Suitable carriers include any material that retains the antitumor function of the therapeutic composition when combined with the therapeutic composition and generally does not react with the patient's immune system. Pharmaceutically acceptable carriers include any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption retarders, and the like. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate, and combinations thereof. Often, the composition contains isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. The pharmaceutically acceptable carrier may further comprise a small amount of auxiliary material, such as a wetting or emulsifying agent, a preservative or a buffer, which enhances the shelf life or effectiveness of the ADC.

The therapeutic formulation may be solubilized and administered by any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic. The therapeutic protein preparation can be lyophilized, stored as a sterile powder, eg, under vacuum, and then reconstituted in bacteriostatic water (eg, containing a benzyl alcohol preservative) or sterile water prior to injection. The therapeutic formulation may include ADC or a pharmaceutically acceptable salt thereof, such as mesylate.

In some embodiments, the ADC is administered to the patient daily, bimonthly, or at any time in between. Dosages and dosing protocols for treating cancer using the aforementioned methods will vary by method and target cancer and will generally depend on a number of other factors recognized in the art.

Various delivery systems are known and can be used for administration of one or more ADCs of the present disclosure. The method of administration of ADC is not limited, but is parenteral administration (for example, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (for example, intranasal administration). And the oral route). In addition, intrapulmonary administration may be used, for example, by the use of an inhaler or nebulizer, and a pharmaceutical product containing an aerosolizing agent. For example, US Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5, 5. , 874,064, 5,855,913, 5,290,540, and 4,880,078; and International Publication No. 1992/09244. Refer to the compositions and methods for intrapulmonary administration described in the Pamphlet, 1997/032572 Pamphlet, 1997/044013 Pamphlet, 1998/031346 Pamphlet, and 1999/06603 Pamphlet. matter. The ADC may be administered by any convenient route, eg, by injection or bolus injection, or by absorption from the epithelial or mucocutaneous lining (eg, oral mucosa, rectum and intestinal mucosa, etc.). The administration may be either systemic administration or local administration.

The therapeutic compositions disclosed herein can be sterile and stable under manufacturing and storage conditions. In some embodiments, the ADC, or one or more of the pharmaceutical compositions, is fed to the hermetically sealed container as a dry sterile lyophilized powder or a water-free concentrate, which is suitable for administration to the subject. It can be reconstituted to a concentration (eg, with water or saline). In some embodiments, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions are at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or. It is supplied to the hermetically sealed container as a dry sterilized lyophilized powder in at least 100 mg, or any amount in between, in a unit dosage. In some embodiments, the lyophilized ADC or pharmaceutical composition is stored in the original container at 2 ° C-8 ° C. In some embodiments, one or more of the ADCs or pharmaceutical compositions described herein are delivered in liquid form to a hermetically sealed container, eg, a container indicating the amount and concentration of the drug. In some embodiments, the liquid form of the composition administered is at least 0.25 mg / mL, at least 0.5 mg / mL, at least 1 mg / mL, at least 2.5 mg / mL, at least 5 mg / mL, at least 8 mg. / ML, at least 10 mg / mL, at least 15 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, or at least 100 mg / mL ADC hermetically sealed vessels. The liquid form may be stored in the original container at 2 ° C to 8 ° C.

In some embodiments, the disclosed ADCs can be incorporated into pharmaceutical compositions suitable for parenteral administration. The solution for injection may consist of either a flint or amber vial, an ampoule, or a prefilled syringe, or a liquid or lyophilized form in another known delivery or storage device.

The compositions described herein may be in various forms. They include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (eg, injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Can be mentioned. The preferred form depends on the intended mode of administration and therapeutic application.

In various embodiments, treatment comprises a single bolus or repeated administration of the ADC preparation by an acceptable route of administration.

Patients may be evaluated for target antigen levels (eg, target antigen-expressing cell levels) in a given sample to aid in determining the most effective dosing regimen and the like. An exemplary embodiment is a method of determining whether treatment with an ADC of the present disclosure is effective for a patient, comprising providing a biological sample from the patient and contacting the ADC with the biological sample. be. Exemplary biological samples include inflammatory exudates, blood, serum, intestinal fluid, stool samples, or tumor biopsies (eg, cancers expressing the target antigen, such as HER2-expressing cancers, CD138-expressing cancers, EPHA2-expressing cancers). Or tissue or body fluid such as a tumor biopsy) derived from a patient at risk thereof. In some embodiments, samples (eg, tissues and / or body fluids) can be obtained from the subject and target antigens (eg, HER2, CD138, EPHA2, MSLN, FOLH1, etc.) can be obtained using suitable immunological methods. Protein expression of CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1) can be detected and / or measured. Such assessments are also used for monitoring throughout the therapy and are useful in combination with assessments of other parameters to determine the success of the therapy.

In some embodiments, the effectiveness of the ADC may be assessed by contacting the ADC with a tumor sample from the subject and assessing the tumor growth rate or volume. In some embodiments, if the ADC is determined to be effective, it can be administered to the subject.

The above treatment techniques can be combined with any one of a wide variety of additional surgery, chemotherapy, or radiation therapy regimens. In some embodiments, the ADC or composition disclosed herein is co-formulated and / or co-administered with one or more additional therapeutic agents, such as one or more chemotherapeutic agents. .. Non-limiting examples of chemotherapeutic agents include alkylating agents such as nitrogen mustards, ethyleneimine compounds, and alkyl sulfonates; antimetabolites such as folic acid, purine or pyrimidine antagonists; antimitotic agents. Anti-tubulin agents such as, for example, eribulin or eribulin mesylate (Halaven ™), bincaalkaloids, and auristatins; cytotoxic antibiotics; damaging or interfering with DNA expression or replication. Compounds include, for example, DNA sub-groove binding agents; and growth factor receptor antagonists. In some embodiments, the chemotherapeutic agent may be a cytotoxic agent or a cell proliferation inhibitor. Examples of cytotoxic agents include, but are not limited to, eribulin or eribulin mesylate (Halaven ™), auristatins (eg, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), Maytan. Anti-thread splitting agents such as cinoids (eg, maytancin), dorastatins, duostatins, cryptophycins, binca alkaloids (eg, bincristin, binblastin), taxans, taxols, and corhitin; anthraculin Classes (eg, daunorbisin, doxorubicin, dihydroxyanthracindione); cytotoxic antibiotics (eg, mitomycins, actinomycins, duocalmycins (eg, CC-1065), auromycins). , Duomycins, Calicaremycins, Endomycins, Phenomycins); Alkylating agents (eg, cisplatin); Intercalating agents (eg, etidium bromide); Topoisomerase inhibitors (eg, etoposides) , Tenoposide); radioactive isotopes such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² or ²¹³ , P ³² , and radioactive isotopes of lutetium (eg,). Lu ¹⁷⁷ ); and toxins of bacterial, fungal, plant or animal origin (eg, lysine (eg, lysine A chain), difteria toxins, Pseudomonas exotoxin A (eg, PE40), endotoxin, mitogellin). , Combrestatin, restrictocin, geronin, α-salcin, abulin (eg, abrin A chain), modesin (eg, modesin A chain), curicin, crotin, sapaonaria officinalis inhibitor, glucocorticoid) Can be mentioned.

Also disclosed herein are, for example, the use of one or more of the disclosed ADCs in the manufacture of therapeutic agents for cancer according to the methods described above. In some embodiments, the ADC disclosed herein is used, for example, in the treatment of cancer according to the methods described above.

In various embodiments, kits for use in the clinical laboratory and therapeutic applications described herein are within the scope of the present disclosure. Such kits are compartmentalized to receive one or more containers such as vials, tubes, etc., each containing one of the separate elements used in the methods disclosed herein. The means of transportation, package, or container being used may be included with a label or package insert containing instructions for use, such as those described herein. The kit may include a container containing the drug portion. The disclosure also provides one or more of ADCs packaged in hermetically sealed containers such as ampoules or sachets indicating the amount of drug, or pharmaceutical compositions thereof.

A kit is a container described above and one or more other containers associated thereto, such as a buffer, a diluent, a filter, a needle, a syringe; a vehicle enumerating the contents, a package, a container, a vial. And / or a container containing materials desirable from a commercial and user standpoint, including tube labels and / or instructions for use, and package inserts containing instructions for use.

Labels may be present on or with the container to indicate that the composition is used for a particular treatment or for non-therapeutic applications such as prognostic, prophylactic, diagnostic, or laboratory applications. Indications may also indicate instructions for use, either in vivo or in vitro, such as those described herein. Instructions and / or other information may also be included in one or more package inserts or one or more indications included with or on the kit. The label may be on or attached to the container. The label may be on the container when the letters, numbers, or other letters forming the label are molded or etched into the container itself. The label may accompany the container, for example as a package insert, in a container or means of delivery that also holds the container. Labeling may indicate that the composition is used in the diagnosis or treatment of conditions such as cancer described herein.

Neoantigens and Methods of Use Also disclosed herein are methods of treating a patient by inducing neoantigens in tumor cells that the patient's immune system can target for clearance in various embodiments. Although not constrained by theory, in various embodiments, administration of a harboxydienspiring regulator alone and / or as part of an ADC or composition induces an immune response, eg, intron resident. Reexpression of the endogenous retrovirus can result in neoantigens that induce a double-stranded RNA immune response and / or neoantigens that induce immunogenic cell death.

As used herein, the term "neoantigen" results from one or more tumor-specific mutations that the immune system has never been exposed to and / or a harboxydiene splicing regulator (eg, eg). Refers to any antigen resulting from tumor exposure to any one or more of the harboxydiene splicing regulators disclosed herein, alone and / or as part of an ADC or composition. Tumor-specific mutations can include missense mutations, frameshifts, translocations, and mRNA splicing variants, as well as mutations that affect post-translational processing such as phosphorylation and glycosylation. These exemplary mutations may, in various embodiments, result from mutations that alter non-synonymous coding changes and / or mRNA processing (eg, splicing). All of these exemplary mutations can, in various embodiments, result in molecular changes that can be discerned by the appropriate T cell receptor. In various preferred embodiments, the exemplary neoantigen is a neoantigen that is induced alone and / or by delivery of a harboxydiene splicing regulator as part of an ADC or composition. In various embodiments, delivery of a harboxydiene splicing regulator (eg, any one or more of the harboxydiene splicing regulators disclosed herein) has never been exposed to the immune system 1 It is possible to induce novel mRNA splicing that results in translation of proteins containing one or more novel peptide domains. In various embodiments, the tumor-specific mutation may be a harboxydiene splicing regulator, an ADC, or an mRNA splicing variant resulting from delivery or administration of a composition comprising a harboxydiene splicing regulator or ADC. ..

Although not constrained by theory, in various embodiments, delivery of the harboxydiene splicing regulator alone and as part of the ADC or composition is an open reading frame and / or of various genes. It can induce novel mRNA splicing (eg, exon skipping, intron retention) that causes changes in the coding sequence. In various embodiments, these altered genes are translated into proteins containing one or more novel peptide domains that the immune system recognizes as exogenous. In various embodiments, these one or more novel peptide domains are not present in the protein without herboxidiene splicing regulatory therapy, or in any other part of the human proteome. In various embodiments, proteins containing one or more novel peptide domains can be degraded by the proteasome to produce novel peptide fragments, which serve as substrates for immune peptide presentation mechanisms, eg, via MHC presentation. .. In various embodiments, the novel peptide fragment corresponding to the neoantigen can be presented, for example, in a MHC1-bound peptide boom on tumor cells.

In various embodiments, delivery of the harboxydiene splicing regulator alone and as part of the ADC or composition can lead to one or more tumor cell specific events (eg, cell growth arrest). In various embodiments, one or more tumor cell-specific events are (1) enhanced association by phagocytic cells (Bracci et al. (2014) Cell Death Differ. 21 (1): 15-25); (2). Association with antigen-presenting cells by transport of novel peptide fragments to tumor influx region lymph nodes; (3) Antigen-presenting cells process new peptide fragments from phagocytosed tumor cells and circulate the fragments as neo-antigens. Presenting to the Naive T cell population; (4) the novel peptide fragment interacts with T cells expressing a receptor that recognizes the fragment as a neoantigen; (5) maturation and activation of the effector T cell response. Further presenting novel peptide fragments corresponding to neoantigens (eg, CD4 + and / or CD8 + T cells; and / or (6) exposed to harboxidien splicing regulator therapy and on its surface MHC1 complex. Can lead to T cell association with tumor cells. In various embodiments, one or more tumor cell specific events, either directly or indirectly, are T cell associations of effector function and / or neo. It can result in the death of tumor cells that present the antigen.

Also, without being bound by theory, in various embodiments, delivery of a harboxydiene splicing regulator alone and as part of an ADC or composition is an intron-resident endogenous retrovirus. It can cause reexpression and lead to a double-stranded RNA immune response.

Furthermore, although not constrained by theory, in various embodiments, delivery of the harboxydiene splicing regulator alone and as part of the ADC or composition is a mutation induced by the splice regulator. It can lead to immunogenic cell death induced by the release of the derived neoantigen. In various embodiments, delivery of the harboxydiene splicing regulator alone and as part of the ADC or composition can induce a double-stranded RNA immune response. In various embodiments, the double-stranded RNA immune response can be caused by the reexpression of the intron-resident endogenous retrovirus. In various embodiments, a double-stranded RNA immune response can result in tumor cell death. In various embodiments, delivery of the harboxydiene splicing regulator alone and as part of the ADC or composition can induce immunogenic cell death. In various embodiments, immunogenic cell death can result from the release of mutation-derived neoantigens and / or host immune responses to tumor cells.

Thus, in various embodiments, compositions comprising one or more harboxydiene splicing regulators and / or ADCs and / or harboxydiene splicing regulators or ADCs, eg, any harbo disclosed herein. Disclosed are therapeutic methods comprising inducing a neo-antigen by administering a xidiene splicing regulator, ADC, or composition. In various embodiments, the method comprises administering a lower dosage of a harboxydiene splicing regulator, ADC, or composition than would have been required without the induction of neoantigen. .. In some embodiments, the method administers one or more initial induction doses to produce neoantigen and induce an immune response (eg, converting naive T cells to memory cells). Subsequent reduced dosage or frequency of dosing (ie, due to the combined effect of the harboxydiensplicing regulator, ADC, or composition with immunotargeting of the neoantigen) is included. In some embodiments, treatment involves administering a harboxydiene splicing regulator, ADC, or composition to induce a neoantigen-based immune response and at least one additional therapy (eg, a second anti). May include combination with cancer therapy). For example, in some embodiments, treatment involves administering a harboxydiene splicing regulator, ADC, or composition to induce a neoantigen-based immune response and one or more checkpoint inhibitors. May include combination. In some embodiments, treatment is the administration of a harboxydiene splicing regulator, ADC, or composition to induce a neoantigen-based immune response in combination with one or more cytokines or cytokine analogs. May include. In some embodiments, treatment comprises administering a harboxydiene splicing regulator, ADC, or composition to induce a neoantigen-based immune response in combination with one or more neoantigen vaccines. obtain. In some other embodiments, the treatment is administration of a harboxydiene splicing regulator, ADC, or composition to induce a neoantigen-based immune response and one or more modified tumor-targeted T cells. May include combination with (eg, CAR-T).

In some embodiments, neoantigens can be used to monitor the effectiveness of treatment with harboxydiene splicing regulators, ADCs, or compositions. For example, patient samples (eg, tumor biopsies) can be obtained after administration of a harboxydiene splicing regulator, ADC, or composition and screened for neoantigens or for identification factors for immune or inflammatory responses. If a neoantigen and / or immune response is detected, further treatment can be provided, eg, at reduced dosages.

In various embodiments, compositions comprising one or more harboxydiene splicing regulators and / or ADCs and / or harboxydiene splicing regulators or ADCs, eg, any harboxydiene disclosed herein. Disclosed are therapeutic methods comprising inducing a double-stranded RNA immune response by administering a splicing regulator, ADC, or composition.

In various embodiments, compositions comprising one or more harboxydiene splicing regulators and / or ADCs and / or harboxydiene splicing regulators or ADCs, eg, any harboxydiene disclosed herein. Disclosed are therapeutic methods comprising inducing immunogenic cell death by administering a splicing regulator, ADC, or composition.

In various embodiments, administration of a harboxydiene splicing regulator, ADC, or composition comprising a harboxydiene splicing regulator can be combined with any known anti-cancer therapy. Examples of immune activation strategies available for current oncology treatments include, but are not limited to, treatment with immune checkpoint inhibitor (ICI) molecules, treatment with cytokines or cytokine analogs, inoculation of tumor-related vaccines, and Modifications of tumor-targeted T cells (eg, enlargement of tumor-infiltrating lymphocytes or CAR-T) can be mentioned. These techniques are primarily focused on enhancing or inducing an immune response against pre-existing tumor antigens (either mutations or aberrant expression of cell surface proteins). One or more of these strategies may involve one or more mutations capable of inducing an antigenic T cell response. For example, patient response to checkpoint inhibition may correlate with non-synonymous mutation loading. In addition, existing mutations and cancer vaccine techniques that rely on the antigenicity of those mutations may be used.

ADCs containing harboxydiene splicing regulators and / or such regulators can induce a wide range of transcriptome changes that occur in multiple systems. Translation of these mRNA changes can result in robust, reproducible protein changes that produce high-affinity MHC1-binding neopeptides across multiple HLA isotypes. Although not constrained by theory, treatment with harboxydiene splicing regulators and / or ADCs potentially enhances the number of responsive neoantigens due to numerous changes in transcriptome and proteome. And can enhance the involvement of adaptive immune responses.

As used herein, the terms "harboxydiene splicing regulator", "spliceosome regulator", "spliceosome regulator", or "spliceosome regulator" interact with components of the spliceosome. This refers to a compound having anticancer activity. In some embodiments, the splicing regulator alters the rate or morphology of splicing in the target cells. For example, harboxydiene splicing regulators that act as inhibitory agents have the ability to reduce uncontrolled cell proliferation. In particular, in some embodiments, the harboxydiene spliceosome regulator may act by inhibiting the SF3b spliceosome complex. In some embodiments, the harboxydiene splicing regulator is selected from any one or more of the harboxydiene splicing regulators disclosed herein. In some embodiments, the harboxydiene splicing regulator is used as a single agent, delivered to cells, and / or administered to a subject. In some other embodiments, the harboxydiene splicing regulator is used as part of an ADC (eg, an ADC selected from any of the exemplary ADCs disclosed herein) and delivered to cells. And / or administered to the subject. In some other embodiments, the harboxydiene splicing regulator is part of a composition comprising multiple copies of the harboxydiene splicing regulator or multiple copies of the ADC carrying the harboxydiene splicing regulator. Used, delivered to cells, and / or administered to a subject. Such compositions are disclosed herein.

In some embodiments, it is used as part of an ADC (eg, an ADC selected from any of the exemplary ADCs disclosed herein), delivered to cells, and / or administered to a subject. Harboxydiene splicing regulators are used as a single drug and provide additional therapeutic benefit compared to harboxydiene splicing regulators that are delivered to cells and / or administered to a subject. For example, in some embodiments, a harboxidiene splicing regulator that is used as part of an ADC, delivered to a cell, and / or administered to a subject is targeted by a target antigen (ie, an antibody portion of the ADC). It results in targeted delivery of the harboxydiene splicing regulator to neoplastic cells expressing the antigen). In some embodiments, such targeted harboxydiene splicing regulatory delivery reduces off-target therapy and / or off-target cytotoxicity. In some embodiments, such targeted delivery enhances tumor-selective neoantigen presentation on neoplastic cells rather than on healthy cells that do not express the target antigen. In some embodiments, such targeted delivery is, for example, at least 75%, 80%, 85%, 90%, 95% of alternative splicing and induction of novel mRNAs and MHC-binding peptides corresponding to neoantigens. , Or 99% lead to what happens in targeted neoplastic cells rather than off-target cells. Thus, although not constrained by theory, in some embodiments, effector T cells because neoantigen is preferentially expressed on tumor cells after treatment with ADC as disclosed herein. After priming and / or expansion (eg, using neoantigen vaccine), the immune system may preferentially attack neoantigen-presenting neoplastic cells rather than healthy cells.

Immune induction and treatment regimen:
In various embodiments, the present disclosure comprises an effective amount of a harboxydiene splicing regulator, a harboxydiene splicing regulator-based antibody-drug conjugate (ADC), or a harboxydiene splicing regulator or ADC. Provides a method of inducing at least one neoantigen by contacting with a neoplastic cell. In various embodiments, the present disclosure comprises an effective amount of a harboxydiene splicing regulator, a harboxydiene splicing regulator-based antibody-drug conjugate (ADC), or a composition comprising a harboxydiene splicing regulator or ADC. Provides a method of inducing a double-stranded RNA immune response by contacting a neoplastic cell. In various embodiments, the present disclosure comprises an effective amount of a harboxydiene splicing regulator, a harboxydiene splicing regulator-based antibody-drug conjugate (ADC), or a harboxydiene splicing regulator or a composition comprising an ADC. Provides a method of inducing immunogenic cell death by contacting with neoplastic cells.

In some embodiments, neoplastic cells are present in in vitro cell cultures. In some embodiments, neoplastic cells are obtained from the subject. In some embodiments, neoplastic cells are present in the subject. In some embodiments, neoplastic cells are derived from hematological malignancies or solid tumors. In some embodiments, the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. In some embodiments, the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, solid tumors are breast cancer (eg, HER2-positive breast cancer), gastric cancer (eg, gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, serous). Endometrial cancer), salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colon-rectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In various embodiments, the present disclosure relates to a composition comprising an effective amount of a harboxydiene splicing regulator, ADC, or harboxydiene splicing regulator or ADC in a subject having or suspected of having a neoplastic disorder. Further provides a method of inducing at least one neoantigen and / or T cell response by administration to. Also described herein are compositions that, in various embodiments, include, in various embodiments, an effective amount of a harboxydiene splicing regulator, an ADC, or a harboxydiene splicing regulator or ADC for a subject having or suspected of having a neoplastic disorder. Methods of treatment by administering the substance to the subject are also provided, wherein administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition induces at least one neoantigen and / or T cell response. do.

In various other embodiments, the present disclosure comprises an effective amount of a harboxydiene splicing regulator, ADC, or harboxydiene splicing regulator or ADC in a subject having or suspected of having a neoplastic disorder. Provided is a method for inducing a double-stranded RNA immune response by administering to a subject. Also described herein are compositions comprising, in various embodiments, an effective amount of a harboxydiene splicing regulator, an ADC, or a harboxydiene splicing regulator or ADC for a subject having or suspected of having a neoplastic disorder. Methods of treatment by administering the substance to a subject are also provided, wherein administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition induces a double-stranded RNA immune response.

Still in other embodiments, the present disclosure comprises an effective amount of a harboxydiene splicing regulator, ADC, or harboxydiene splicing regulator or ADC in a subject having or suspected of having a neoplastic disorder. Provided is a method for inducing immunogenic cell death by administering to a subject. Further, in various embodiments, the present specification comprises an effective amount of a harboxydiene splicing regulator, an ADC, or a harboxydiene splicing regulator or ADC for a subject having or suspected of having a neoplastic disorder. A method of treatment is provided by administering a substance to a subject, wherein administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition induces immunogenic cell death.

In some embodiments, the present disclosure presents an effective amount of a harboxydiene splicing regulator in combination with one or more additional therapies, including a second agent, in a subject with or suspected of having a neoplastic disorder. , ADC, or harboxydiene splicing regulators or compositions comprising ADCs to further provide a method of treatment, wherein the harboxydiene splicing regulators, antibody-drug conjugates, or compositions. Administration induces immunogenic cell death.

In some embodiments of the therapeutic methods described herein, the dosed amount of a harboxydienplising regulator, antibody-drug conjugate, composition, or second agent is harboxydiensplicing. The induction of at least one neoantigen and / or T cell response is reduced when compared to standard dosages of regulatory agents, antibody-drug conjugates, compositions, or second agents. In some embodiments, the dose of the harboxydiene splicing regulator, antibody-drug conjugate, composition, or second agent is the harboxydiene splicing regulator, antibody-drug conjugate, composition, or. Reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to the standard dosage of the second drug. To. In some embodiments, the harboxydiene splicing regulator, antibody-drug conjugate, composition, or second agent is a harboxydiene splicing regulator, antibody-drug conjugate, composition, or second agent. Administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently when compared to the standard dosing regimen of the drug. Will be done. In some embodiments, the dosage and / or dosage of the harboxydiene splicing regulator, antibody-drug conjugate, composition, or second agent reduces systemic toxicity and / or improves tolerability. Bring.

As used herein, the term "standard dosage" or "standard dosing regimen" refers to any conventional or routine dosing regimen for a therapeutic agent, eg, a regimen proposed by the manufacturer. Refers to a regimen approved by a regulatory agency, or otherwise tested in humans to meet the needs of the average patient. In some embodiments, the therapeutic agent is a harboxydiene splicing regulator, antibody, or antibody-drug conjugate with anti-cancer activity.

For example, the standard dosing regimen for trastuzumab, an exemplary anti-HER2 antibody disclosed herein, is 8 mg / kg (week 1) given intravenously over 90 minutes, followed by every 3 weeks. It may be 6 mg / kg (from week 4 to the end of the therapy cycle) administered intravenously over 30-90 minutes (Herceptin® (Trastuzumab) FDA Labeling Supplement, 2017).

As another example, the standard dosing regimen of ipilimumab, an exemplary anti-CTLA4 checkpoint inhibitor antibody, may be 3 mg / kg given intravenously over 90 minutes every 3 weeks over 4 doses (4 doses). Yervoy® (ipilimumab) FDA Labeling Supplement, 2018). Another standard dosing regimen for ipilimumab may be 10 mg / kg intravenously over 90 minutes every 3 weeks over 4 doses, followed by 10 mg / kg every 12 weeks for up to 3 years. (Yervoy® (Ipilimumab) FDA Labeling Supplement, 2018).

As another example, the standard dosing regimen of nivolumab, an exemplary anti-PD1 checkpoint inhibitor antibody, may be 3 mg / kg given intravenously over 60 minutes every two weeks (Opdivo (Registration). Trademark) (nivolumab) FDA indication, 2015).

As another example, the standard dosing regimen for the exemplary anti-PDL1 checkpoint inhibitor antibody, atezolizumab, may be 1200 mg intravenously over 60 minutes every 3 weeks (Tekentriq®). (Atezolizumab) FDA Labeling Supplement, 2018).

As yet another example, the standard dosing regimen for the exemplary anti-HER2 antibody-drug conjugate T-DM1 may be 3.6 mg / kg given intravenously over 90 minutes every 3 weeks. Good (Kadcyla® (T-DM1) FDA Labeling Supplement, 2016).

In some embodiments, the methods described herein are to administer at least one additional therapy (eg, checkpoint inhibitor, neoantigen vaccine, cytokine or cytokine analog, CAR-T, etc.). Further may be included. In some embodiments, the dosed dose of a harboxydiensplicating regulator, antibody-drug conjugate, composition, and / or at least one additional therapy is a harboxydienspiring regulator, antibody-drug conjugate. , Composition, and / or the induction of at least one neoantigen and / or T cell response when compared to the standard dosage of at least one additional therapy. In some embodiments, the dosed dose of a harboxydiensplicating regulator, antibody-drug conjugate, composition, and / or at least one additional therapy is a harboxydienspiring regulator, antibody-drug conjugate. , Composition, and / or is reduced because of the induction of a double-stranded RNA immune response when compared to standard dosages of at least one additional therapy. In some embodiments, the dosed dose of a harboxydien Splicing regulator, antibody-drug conjugate, composition, and / or at least one additional therapy is a harboxydiensplicing regulator, antibody-drug conjugate. , Composition, and / or reduced because of the induction of immunogenic cell death when compared to standard dosages of at least one additional therapy. In some embodiments, the dose of a harboxydiene splicing regulator, antibody-drug conjugate, composition, and / or at least one additional therapy is a harboxydiene splicing regulator, antibody-drug conjugate, composition. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75% when compared to the standard dosage of the drug and / or at least one additional therapy. , Or 90% reduction. In some embodiments, the harboxydien Splicing regulator, antibody-drug conjugate, composition, and / or at least one additional therapy is a harboxydiensplicing regulator, antibody-drug conjugate, composition, and. / Or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or when compared to the standard dosing regimen of at least one additional therapy. It is administered 90% less frequently. In some embodiments, the dose and / or dosage of the harboxydiene splicing regulator, antibody-drug conjugate, composition, and / or at least one additional therapy reduces systemic toxicity and / or is tolerable. Brings improvement in sex.

In some embodiments, administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is initiated prior to administration of at least one additional therapy. In other embodiments, administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition is initiated after administration of at least one additional therapy. Still in other embodiments, administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is initiated at the same time as administration of at least one additional therapy.

In some embodiments, administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is repeated at least once after the initial dose. In some embodiments, the amount used for repeated doses of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is reduced when compared to the amount used for the initial dose. In some embodiments, the amount used for repeated administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition is a harboxydiene splicing regulator, antibody-drug conjugate, or composition. Reduced when compared to standard dosages. In some embodiments, the amount used for repeated administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition is a harboxydiene splicing regulator, antibody-drug conjugate, or composition. Reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to standard or initial dosages ..

In some embodiments, administration of at least one additional therapy is repeated at least once after the initial administration. In some embodiments, the amount used for repeated doses of at least one additional therapy is reduced when compared to the amount used for the initial dose. In some embodiments, the amount used for repeated doses of at least one additional therapy is reduced when compared to the standard dosage of at least one additional therapy. In some embodiments, the amount used for repeated doses of at least one additional therapy is 10%, 15%, 20% when compared to the standard or initial dosage of at least one additional therapy. , 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% reduction.

In some embodiments, repeated doses of harboxydiene splicing regulators, antibody-drug conjugates, or compositions are simultaneous with repeated doses of at least one additional therapy. In some embodiments, administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is sequential or staggered with repeated doses of at least one additional therapy.

In some embodiments, at least one additional therapy comprises administering a checkpoint inhibitor, eg, any checkpoint inhibitor disclosed herein. In some embodiments, the subject is intolerant, intolerant, or refractory to the checkpoint inhibitor when administered alone. In some embodiments, the checkpoint inhibitor targets PD1 / PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and / or KIR. In some embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR. In some embodiments, the checkpoint inhibitor is an antibody that has inhibitory or agonistic activity against its target. In some embodiments, the checkpoint inhibitor is targeted with an inhibitory antibody or other similar inhibitory molecule. In other embodiments, the checkpoint inhibitor is targeted with an agonist antibody or other similar agonist molecule.

In some other embodiments, at least one additional therapy comprises administering a neoantigen vaccine, eg, any neoantigen vaccine disclosed herein. In some embodiments, the harboxydiene splicing regulator, ADC, or composition is administered prior to administration of the neoantigen vaccine. In some embodiments, the harboxydiene splicing regulator, ADC, or composition is administered after administration of the neoantigen vaccine. In some embodiments, the harboxydiene splicing regulator, ADC, or composition is administered at the same time as the administration of the neoantigen vaccine. In some embodiments, administration of the harboxydiene splicing regulator, ADC, or composition is repeated at least once after the initial dose. In some embodiments, the amount used for repeated doses of the harboxydiene splicing regulator, ADC, or composition is reduced when compared to the amount used for the initial dose.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length. In some embodiments, the at least one neoantigen peptide comprises one or more neoantigen sequences.

In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety does not exclusively overlap or overlap with the canonical peptide sequence (eg, any of the exemplary canonical peptide sequences underlined in Table 8). Then it doesn't.

The term "antigenic portion" or "antigenic fragment" of a neo-antigen sequence, as used herein, refers to a T cell response (eg, antigen-specific expansion of one or more effector T cell populations and / Or refers to one or more fragments of a neoantigen sequence that retains the ability to induce maturation). The antigenic moiety may also retain the ability to be internalized, processed, and / or presented by antigen presenting cells (eg, dendritic cells) in some embodiments. In some embodiments, the antigenic moiety also retains T cell priming function. In some embodiments, the antigenic portion of the neoantigen sequence ranges from about 10 to about 50 amino acids in length. In some embodiments, the antigenic portion of the neoantigen sequence ranges from about 10 to about 35 amino acids in length. In some embodiments, the antigenic portion of the neoantigen sequence ranges from about 15 to about 25 amino acids in length. In some embodiments, the antigenic portion of the neoantigen sequence ranges from about 10 to about 20 amino acids in length. In some embodiments, the antigenic portion of the neo-antigen sequence (eg, the antigenic portion of any one of SEQ ID NOs: 66-93), or its coding mRNA, is formulated as a neo-antigen vaccine.

An exemplary embodiment of the antigenic moiety is one or more regions flanking amino acids 45-53 of SEQ ID NO: 66. Another exemplary embodiment of the antigenic moiety is one or more regions flanking amino acids 82-90 of SEQ ID NO: 66. In some embodiments, the antigenic moiety has the ability to bind to at least one HLA allele expressed in the subject (eg, HLA-A * 02: 01). In some other embodiments, the antigenic moiety is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 of the subjects in the subject population suffering from neoplastic disorders. Has the ability to bind to at least one HLA allele expressed in%, at least 40%, or at least 45%. In some embodiments, the antigenic moiety has the ability to elicit a T cell response to tumors present in at least 1%, at least 5%, or at least 10% of the subject population suffering from neoplastic disorders.

In some embodiments, the antigenic moiety does not, or consists exclusively of, the canonical peptide sequence. The term "canonical peptide sequence", as used herein, has no contact with a harboxydiene splicing regulator (eg, harboxydiene alone and / or as part of an ADC or composition. Refers to any contiguous peptide sequence present in the human proteome (without contact with the splicing regulator) and / or having been exposed to immunity in the past. In some embodiments, the canonical peptide sequence is derived from and / or encoded by the canonical transcript open reading frame. The exemplary canonical peptide sequences are underlined in Table 8.

In some embodiments, when the harboxydiene splicing regulator is administered (eg, alone and / or as part of an ADC or composition), the canonical peptide sequence is induced by the harboxydiene splicing regulator. It can be derived from and / or encoded by the inframe 24 nucleotides on the 5'side immediately prior to the anomalous splicing event. Thus, in some embodiments, the canonical peptide sequence comprises or consists of 8 amino acids immediately N-terminal to the neoantigen sequence induced by the harboxidien splicing regulator. In some embodiments, when the 5'exon sequence terminates at a codon-terminated nucleotide, the canonical peptide sequence terminates at the end of that exon. In some other embodiments, when the 5'exon sequence terminates with one or two of the three nucleotides of the codon, the canonical peptide sequence is derived from the 24 nucleotides preceding the defective codon. , And / or coded thereby. In some embodiments, the mRNA sequence on the 3'side of the abnormal splicing event can be translated within the same open reading frame from the 5'exon until a stop codon at which translation can be terminated is reached. In some embodiments, when the canonical transcript open reading frame is conserved as a result of an abnormal splicing event (eg, exon skipping), the C-terminal sequence is translated with an additional 24 nucleotides encoding the C-terminal 8 amino acids. obtain. In this regard, in some embodiments, only the region spanning the aberrant exon junction may encode the neoantigen sequence. In some embodiments, when the open reading frame shifts (eg, intron retention), the complete C-terminal sequence (encoded by 3'mRNA) may encode the neoantigen sequence.

In some embodiments, the antigenic portion of the neoantigen sequence compares the neoantigen sequence with the canonical peptide sequence; and does not exclusively overlap, consists of, and / or does not align with the canonical peptide sequence. It is selected by selecting a part of the array. The antigenic portion of the neo-antigen sequence, in some embodiments, is similar to the full-length neo-antigen sequence (eg, the neo-antigen sequence from which the antigenic moiety is derived) with respect to antigenicity and / or T cell priming function. Can be screened. In some embodiments, the antigenic portion of the neoantigen sequence is assessed for antigenicity and / or T cell priming function using a T cell priming assay, such as the exemplary T cell priming experiments described herein. To.

In some embodiments, the neoantigen sequence is a subject-specific neoantigen sequence. In some embodiments, the neoantigen sequence is a neoantigen vaccine personalized for the subject. In some embodiments, the neoantigen sequence used to create a neoantigen vaccine personalized for a subject has the ability to bind at least one HLA allele expressed in the subject. In some embodiments, the personalized neo-antigen vaccine identifies the neo-antigen expressed in the tumor of interest, eg, after administration of a harboxidien splicing regulator or ADC, and the neo-antigen found in the patient's tumor. It is selected by selecting the vaccine containing the sequence.

The term "individualized", when used in the description of a neo-antigen vaccine, to one or more neo-antigens produced in a patient, preferably to a harboxidien splicing regulator, ADC, or composition in the patient. Refers to a vaccine made by identifying what is identified after exposure to and then using one or more of those neoantigens as the basis for a vaccine for the same patient. Therefore, in some embodiments, the patient is given a harboxydiene splicing regulator, ADC, or composition and screened for the neoantigen produced by the treatment. In some embodiments, the neoantigen vaccine of choice is disclosed herein and confirmed to be present in the patient after exposure to a harboxydiene splicing regulator, ADC, or composition. Contains antigenic peptides or mRNA. In some embodiments, the harboxydiene splicing regulator, ADC, or composition and / or peptide or mRNA vaccine may be administered to the patient once or repeatedly. Then, in some embodiments, one or more of those neoantigens are used to create a personalized vaccine to be given to the patient. In some embodiments, the one or more neoantigens used to create a personalized vaccine have a binding affinity for one or more patient-specific HLA alleles. In some embodiments, the patient expresses one or more MHC1 alleles that bind to one or more neoantigens. Prediction of whether a given neoantigen binds to a specific MHC1 allele can be determined using any computational prediction method known in the art. For exemplary computational prediction methods, see, for example, Maydan et al. (2013) BMC Bioinformatics 14 (Suppl. 2): S13, which is incorporated herein by reference for such methods.

In some other embodiments, the neoantigen sequence is a universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal neoantigen vaccine.

The term "universal", when used in the description of a neoantigen vaccine, is preferably produced in multiple patients and / or patient tissue samples after exposure to a harboxidien splicing regulator, ADC, or composition. Refers to a vaccine having a common or known neoantigen-based peptide or mRNA sequence found by sequencing neoantigens. The peptide or mRNA sequence used in the vaccine does not have to be present in every patient, but rather it is sufficient to be found in at least some patient or patient tissue samples. In some embodiments, the harboxydiene splicing regulator, ADC, or composition and / or peptide or mRNA vaccine may be administered to the patient once or repeatedly. Then, in some embodiments, the peptide or mRNA sequence in question is used for further patient vaccination. In some embodiments, the patient is given a harboxydienplising regulator, ADC, or composition, followed by a known neoantigen peptide or mRNA vaccine, thereby giving the harboxydiensplicing regulator, ADC, or composition. Enhances the immune response to the neoantigen produced by the composition. In some embodiments, the patient is given a universal peptide or mRNA vaccine, followed by a harboxydiene splicing regulator, ADC, or composition once or repeatedly. In some embodiments, one or more neoantigen sequences used to create a universal neoantigen vaccine are selected based on the overall MHC1 allele frequency in a given patient population (Maiers et al. 2007) Hum. Immunol. 68 (9): 779-88).

In some embodiments, neoantigen (eg, universal neoantigen) sequences are at least 10%, at least 15%, at least 20%, at least 25%, at least of subjects in a population of subjects suffering from neoplastic disorders. It has the ability to bind to at least one HLA allele expressed in 30%, at least 35%, at least 40%, or at least 45%. In some embodiments, the neoantigen sequence has the ability to elicit a T cell response to tumors present in at least 1%, at least 5%, or at least 10% of the subject population suffering from neoplastic disorders.

In some embodiments, the neoantigen sequence is at least one neoantigen peptide induced in a subject by administration of an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition, or a combination thereof. It has been identified by sequencing the coding mRNA. In some embodiments, the at least one neoantigen peptide provides a neoantigen sequence derived by contacting an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition with neoplastic cells. include. In some embodiments, neoplastic cells are present in in vitro cell cultures. In some embodiments, neoplastic cells are obtained from the subject. In some embodiments, neoplastic cells are present in the subject.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier (eg, any of the exemplary carriers described herein). In some embodiments, the at least one neoantigen peptide is linked to a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. Toxoid. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are covalently linked via a linker. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as fusion proteins. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable adjuvant.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA. In some embodiments, at least one neoantigen mRNA encodes one or more neoantigen sequences.

In some embodiments, the neoantigen sequence is a subject-specific neoantigen sequence. In some embodiments, the neoantigen sequence is a neoantigen vaccine personalized for the subject. In some embodiments, the neoantigen sequence has the ability to bind to at least one HLA allele expressed in the subject.

In some other embodiments, the neoantigen sequence is a universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal neoantigen vaccine. In some embodiments, the neoantigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% of subjects in a population of subjects suffering from neoplastic disorders. It has the ability to bind to at least one HLA allele that is expressed in at least 40%, or at least 45%. In some embodiments, the neoantigen sequence has the ability to elicit a T cell response to tumors present in at least 1%, at least 5%, or at least 10% of the subject population suffering from neoplastic disorders.

In some embodiments, the neoantigen sequence has been identified by sequencing the protein sequence of at least one neoantigen. In some embodiments, the neoantigen sequence is at least one mRNA encoding a neoantigen induced in the subject by administering an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition. Have been identified by sequencing. In some embodiments, the at least one neoantigen mRNA provides a neoantigen sequence derived by contacting an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition with neoplastic cells. Code. In some embodiments, neoplastic cells are present in in vitro cell cultures. In some embodiments, neoplastic cells are obtained from the subject. In some embodiments, neoplastic cells are present in the subject.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier (eg, any of the exemplary carriers described herein). In some embodiments, at least one neoantigen mRNA is ligated to a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. Toxoid. In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable adjuvant. In some embodiments, the neoantigen mRNA is encapsulated in an encapsulant. In some embodiments, the encapsulant is a liposome. In some embodiments, the encapsulant is nanoparticles.

In some embodiments, at least one additional therapy comprises administering a cytokine or cytokine analog, eg, any cytokine or cytokine analog disclosed herein. In some embodiments, the subject is intolerant, intolerant, or refractory to cytokines or cytokine analogs when administered alone. In some embodiments, the cytokine or cytokine analog comprises a T cell enhancer. In some embodiments, cytokines or cytokine analogs include IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα. In some embodiments, cytokines or cytokine analogs include IL-2, IL-10, IL-12, and / or IL-15. In some embodiments, administration of a cytokine or cytokine analog results in T cell priming after administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition due to the induction and presentation of neoantigen. Is enhanced.

In some embodiments, at least one additional therapy comprises administering a modified tumor-targeted T cell (ie, CAR-T), eg, any CAR-T therapy disclosed herein.

In some embodiments, the methods described herein are one or more neoantigen and / or T cell responses in a subject after administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition. And optionally, if one or more neoantigens and / or T cell responses are detected, continue administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition. Can further include that. In some embodiments, detection of one or more neoantigens and / or T cell responses in a subject dictates the efficacy of treatment with a harboxydiene splicing regulator, antibody-drug conjugate, or composition. .. In some embodiments, if one or more neoantigens and / or T cell responses are detected, treatment with additional therapy in combination with a harboxydiene splicing regulator, antibody-drug conjugate, or composition Will be continued. In some embodiments, if one or more neoantigens and / or T cell responses are detected, treatment is continued with reduced dosage and / or frequency.

In some embodiments, the methods described herein are to detect a double-stranded RNA immune response in a subject after administration of a harboxydienspiring regulator, antibody-drug conjugate, or composition. , Optionally, if a double-stranded RNA immune response is detected, may further comprise continuing administration of a harboxidien splicing regulator, antibody-drug conjugate, or composition. In some embodiments, detection of a double-stranded RNA immune response in a subject dictates the efficacy of treatment with a harboxydiene splicing regulator, antibody-drug conjugate, or composition. In some embodiments, if a double-stranded RNA immune response is detected, treatment with additional therapy is continued in conjunction with a harboxydiene splicing regulator, antibody-drug conjugate, or composition. In some embodiments, if a double-stranded RNA immune response is detected, treatment is continued with reduced dosage and / or frequency.

In some embodiments, the methods described herein are to detect immunogenic cell death in a subject after administration of a harboxydienspiring regulator, antibody-drug conjugate, or composition. Optionally, if immunogenic cell death is detected, continued administration of the harboxidien splicing regulator, antibody-drug conjugate, or composition may further be included. In some embodiments, detection of immunogenic cell death in a subject dictates the efficacy of treatment with a harboxydiene splicing regulator, antibody-drug conjugate, or composition. In some embodiments, if immunogenic cell death is detected, treatment with additional therapy is continued in conjunction with a harboxydiene splicing regulator, antibody-drug conjugate, or composition. In some embodiments, if immunogenic cell death is detected, treatment is continued with reduced dosage and / or frequency.

In some embodiments, the subject has a non-synonymous mutation load of about 150 or less mutations. In some embodiments, the subject has a non-synonymous mutation load of about 100 or less mutations. In some embodiments, the subject has a non-synonymous mutation load of about 50 or less mutations. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematological malignancies or solid tumors. In some embodiments, the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. In some embodiments, the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, solid tumors include breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary tract cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and Selected from esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In various embodiments, the present disclosure is a method of treating a subject who has or is suspected of having a neoplastic disorder, wherein (a) an effective amount of a harboxydiensplying regulator, ADC, or harboxydiensplicating regulator. By administering to a subject a composition comprising a drug or ADC, administration of a harboxidien splicing regulator, antibody-drug conjugate, or composition induces at least one neoantigen and / or T cell response. (B) Detecting one or more neoantigens and / or T cell responses in a subject after administration of a harboxydienspiring regulator, antibody-drug conjugate, or composition; and (c) one or more. If a neoantigen and / or T cell response is detected, further provides a method comprising continuing administration of a harboxidien splicing regulator, antibody-drug conjugate, or composition. In some embodiments, detection of one or more neoantigens and / or T cell responses in a subject dictates the efficacy of treatment with a harboxydiene splicing regulator, antibody-drug conjugate, or composition. .. In some embodiments, the one or more neoantigens comprises the amino acid sequence of any one of SEQ ID NOs: 37-65. In some embodiments, the one or more neoantigens comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the one or more neoantigens comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the one or more neoantigens comprises the amino acid sequence of any one of SEQ ID NOs: 46-49.

Harboxydiene splicing regulator / ADC in combination with immune checkpoint inhibition:
In various embodiments, patients with cancer as described herein can be treated with a harboxydiene splicing regulator, ADC, or composition in combination with checkpoint inhibitor therapy. Immune checkpoints are suppressive pathways that slow or slow the immune response to prevent excessive tissue damage due to uncontrolled activity of immune cells. As used herein, the term "checkpoint inhibitor" is any small molecule chemical that inhibits one or more of these inhibitory pathways, thereby allowing for a wider range of immune activity. It is intended to refer to any therapeutic agent, including compounds, antibodies, nucleic acid molecules, or polypeptides, or any fragment thereof.

Treatment of patients with immune checkpoint inhibition has been shown to have robust efficacy for certain clinical indications. Recently, the FDA has approved the use of checkpoint inhibitors in patients with tumors that are tissue-line independent and exhibit high microsatellite instability. This approval was partly based on the observation that response rate was positively correlated with mutation loading (Rizvi et al. (2015) Science 348 (6230): 124-8; Hellmann et. al. (2018) Cancer Cell 33 (5): 853-861). Estimates from the literature suggest that the probability of response increases above the threshold of about 150-250 mutations, although it varies in absolute numbers and from line to line. Analysis of TCGA data shows that the majority of adult-onset tumor lines have relatively low non-synonymous mutation loads (Vogelstein et al. (2013) Science 339: 1549-58). For most strains, the median non-synonymous mutation rate is approximately 30-80 per patient, well below the threshold at which the likelihood of response to checkpoint inhibitors is improved.

For example, HER2-positive breast cancer has been shown to have a median number of non-synonymous mutations present per patient sample of approximately 60. However, as mentioned above, the therapeutic efficacy threshold of checkpoint inhibitors is estimated to be in the range of about 150-250 non-synonymous mutations, i.e., patients above this threshold are in complete remission, partial remission, and partial remission. / Or is likely to exhibit disease stability, while patients below this threshold are likely to exhibit disease progression. Therefore, a strategy to increase the apparent non-synonymous mutation number and / or neoantigen number presented on tumor cells may increase the overall response probability of, for example, checkpoint inhibitor therapy. Since cytokines (and their analogs) work by a similar mechanism of action, such strategies can also increase the overall probability of response to cytokine-based therapies.

The current response rate in HER2-positive breast cancer is approximately 15-25% (CTI NCT02129556). In various embodiments disclosed herein, treatment with a harboxydiene splicing regulator, ADC, or composition in combination with a checkpoint inhibitor and / or cytokine therapy may improve such response rates. .. In various embodiments, treatment with a harboxydiensplying regulator, ADC, or composition in combination with a checkpoint inhibitor and / or cytokine therapy has a median non-synonymous mutation rate of any adult-onset tumor. It can be applied to those below the threshold of an estimated about 150 mutations. In various embodiments, it is suitable for treatment with the harboxydienspiring regulators, ADCs, or compositions of the present disclosure, either alone or in combination with additional therapies (eg, checkpoint inhibitor therapy, cytokine therapy). Exemplary cancer types include, but are not limited to, esophageal cancer, non-Hojikin lymphoma, colorectal cancer, head and neck cancer, gastric cancer, endometrial cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, hepatocellular carcinoma, collagen bud. Examples include tumors, breast cancers (eg, HER2-positive breast cancers), lung cancers (eg, non-small cell lung cancers), chronic lymphocytic leukemia, and acute myeloid leukemia. Other exemplary suitable cancer types are, for example, Vogelstein et al. (2013) Science 339: 1549-58, which is incorporated herein by reference in its entirety.

As many checkpoint inhibitor therapies are based on chronic tumor-related antigen expression, regular therapeutic boosts are needed for efficacy and for "reboost" of responsive T cell populations. The inducible nature of the harboxydiensplicing regulators or ADC-derived neoantigens described herein suppresses T cell depletion, which is often caused by chronic antigen stimulation, while immunizing neoantigen-responsive T cells. A therapeutic dosing regimen that can be designed to enhance the response is provided. For example, in some embodiments, the initial dose of a harboxydiene splicing regulator, ADC, or composition is administered to the subject to induce aberrant splicing and neoantigen peptide production. After sufficient time for protein production and antigen presentation, in some embodiments, the subject is then administered an initial dose of a checkpoint inhibitor to boost and / or enhance effector T cell priming and expansion. Let me. In some embodiments, the waiting period between doses of a harboxydiene splicing regulator, ADC, or composition with a checkpoint inhibitor is about 2, about 3, about 4, about 5, about 6, or about. It's 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR. In some embodiments, the benefits of combination therapy with a harboxydiene splicing regulator, ADC, or composition with a checkpoint inhibitor can be additive or hyperadditive.

In some embodiments, after a period sufficient for T cell priming and expansion, the subject is then administered a second or subsequent dose of a harboxydiene splicing regulator, ADC, or composition. Induces representation of neoantigen peptides. In some embodiments, the waiting period between the initial dose of the checkpoint inhibitor and the second or subsequent dose of the harboxydiene splicing regulator, ADC, or composition is about 2, about 3, About 4 or about 5 weeks. In some embodiments, the waiting period is about 3 weeks. Upon receiving a second or subsequent dose of a harboxidien splicing regulator, ADC, or composition, in some embodiments, the immune system associates with neoantigen-presenting tumor cells and / or causes tumor cell killing. Can trigger. In some embodiments, after allowing secondary T cell priming and expansion, the subject is then administered a second or subsequent dose of checkpoint inhibitor to further expand the memory effector T cell population. To.

In some embodiments, the administration of a harboxydiene splicing regulator, ADC, or composition following this exemplary initial treatment regimen can be pulsed, ie, a harboxydienspiring regulator, ADC, or. The composition is at long enough intervals for antigen presentation, T cell association and / or tumor cell killing, and / or memory T cell population recovery (eg, about every 4 weeks, about every 5 weeks, about every 6 weeks). ) May be administered. At a later time, in some embodiments, the harboxydiene splicing regulator, ADC, or composition therapy is one or more checkpoint inhibitors that target the restoration of effector function to the depleted T cell population. May be used in combination with. For example, in some embodiments, at a later point in time, the harboxydiene splicing regulator, ADC, or composition therapy will inhibit one or more checkpoints targeting PD1 / PDL1, LAG3, and / or TIM3. It may be used in combination with a drug. In some embodiments, the pulsed nature of neoantigen presentation and priming allows low frequency and / or low doses of checkpoint inhibitors and / or harboxydiene splicing regulators, ADCs, or compositions. It may be possible to administer. In some embodiments, the pulsed nature of neoantigen presentation allows, for example, to be compared to a checkpoint inhibitor when administered without concomitant harboxidien splicing regulators, ADCs, or composition treatments. One or more treatments with checkpoint inhibitors (eg, anti-CTLA4 antibodies such as ipilimumab) by reducing the potential risk of adverse reactions often observed with standard dosing regimens of checkpoint inhibitors. Benefits can be brought.

In certain embodiments, the checkpoint inhibitor is an inhibitor of the cytotoxic T lymphocyte-related antigen (CTLA4) pathway. CTLA4, also known as CD152, is a protein receptor that downregulates the immune response. CTLA4 is constitutively expressed on regulatory T cells, but is upregulated only after activation in conventional T cells. As used herein, the term "CTLA4 inhibitor" is intended to refer to any inhibitor of the CTLA4 and / or CTLA4 pathway. Exemplary CTLA4 inhibitors include, but are not limited to, anti-CTLA4 antibodies. CTLA4 blocking antibodies for use in humans have been developed based on the preclinical activity found in mouse models of anti-tumor immunity. Exemplary anti-CTLA4 antibodies include, but are not limited to, ipilimumab (MDX-010) and tremelimumab (CP-675,206), both of which are fully human. Ipilimumab is IgG1 with a plasma half-life of about 12-14 days; tremelimumab is IgG2 with a plasma half-life of about 22 days. For example, Phan et al. (2003) Proc Natl Acad Sci USA. 100: 8372-7; Ribas et al. (2005) J Clin Oncol. 23: 8968-77; Weber et al. (2008) J Clin Oncol. 26: 5950-6. In some embodiments, the anti-CTLA4 antibody is ipilimumab.

In certain embodiments, the checkpoint inhibitor is an inhibitor of the Programmed Death-1 (PD1) pathway. The programmed cell death 1 (PD1) pathway corresponds to the major immune control switch that tumor cells can associate with to survive active T cell immune surveillance. Ligands of PD1 (PDL1 and PDL2) can be constitutively expressed or induced in various tumors. High expression of PDL1 (and to a lesser extent PDL2) on tumor cells has been shown to correlate with prognosis and poor survival in a variety of other solid tumor types. Furthermore, PD1 has been suggested to regulate tumor-specific T cell expansion in patients with malignant melanoma. These observations suggest that the PD1 / PDL1 pathway plays a decisive role in tumor immune avoidance and may be considered an attractive target for therapeutic intervention. As used herein, the term "PD1 inhibitor" is intended to refer to any inhibitor of the PD1 and / or PD1 pathway. Exemplary PD1 inhibitors include, but are not limited to, anti-PD1 and anti-PDL1 antibodies. In certain embodiments, the checkpoint inhibitor is an anti-PD1 antibody. Exemplary anti-PD1 antibodies include, but are not limited to, nivolumab and pembrolizumab (MK-3475). For example, nivolumab is a fully human immunoglobulin G4 (IgG4) PD1 immune checkpoint inhibitor antibody that disrupts the interaction of the PD1 receptor with its ligands PDL1 and PDL2, thereby suppressing the cell-mediated immune response (Guo et). al. (2017) J Cancer 8 (3): 410-6). In some embodiments, the anti-PD1 antibody is nivolumab. For example, pembrolizumab is a strong and highly selective humanized mAb of the IgG4 / κ isotype designed to directly block the interaction between PD1 and its ligands PDL1 and PDL2. Pembrolizumab strongly enhances the T lymphocyte immune response in cultured blood cells from healthy human donors, cancer patients, and primates. Pembrolizumab has also been reported to regulate levels of interleukin-2 (IL-2), tumor necrosis factor α (TNFα), interferon gamma (IFNγ), and other cytokines. Exemplary anti-PDL1 antibodies include, but are not limited to, atezolizumab, avelumab, and durvalumab. For example, atezolizumab is an IgG1 humanized mAb that has been reported to block PD1 / PDL1 interactions by targeting PDL1 expressed on numerous types of malignant cells. This blockade of the PD1 / PDL1 pathway can stimulate anti-tumor immune defense mechanisms (Abdin et al. (2018) Cancers (Basel) 10 (2): 32). In some embodiments, the anti-PDL1 antibody is atezolizumab.

In certain embodiments, the checkpoint inhibitor targets PD1 / PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and / or KIR. In certain embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR. In certain embodiments, the checkpoint inhibitor is targeted with an inhibitory antibody or other similar inhibitory molecule (eg, an inhibitory anti-CTLA4 or anti-PD1 / PDL1 antibody). In certain other embodiments, the checkpoint inhibitor is targeted with a target agonist; examples of this class include stimulatory targets OX40, CD40, and / or GITR. In some embodiments, the checkpoint inhibitor that targets OX40, CD40, and / or GITR is an agonist antibody. Agonist antibodies directed to OX40 may have a dual role of inhibiting regulatory T cell suppression and, on the other hand, enhancing effector T cell function. Agonist anti-GITR antibodies have also been shown to increase the resistance of effector T cells to regulatory T cell-induced inhibition (Karaki et al. (2016) Vaccines (Basel) 4 (4): 37). .. Similarly, the agonist CD40 antibody also demonstrates T cell-dependent antitumor activity. Activation of CD40 on dendritic cells increases cross-presentation of tumor antigens, resulting in an increase in the number of activated tumor-targeting effector T cells (Ellmark et al. (2015) Oncoimmul. 4 (7)). : E1011484).

In certain embodiments, the checkpoint inhibitor targets CTLA4 (eg, anti-CTLA4 antibody). In certain embodiments, targeting CTLA4 promotes priming and activation of naive T cells. In certain embodiments, the checkpoint inhibitor targets OX40 (eg, an anti-OX40 antibody). In certain embodiments, targeting OX40 enhances effector T cell expansion. In certain embodiments, the checkpoint inhibitor targets CD40 (eg, anti-CD40 antibody). In certain embodiments, targeting CD40 inhibits "tolerant" priming of T cells and / or the formation of regulatory T cells. In certain embodiments, the checkpoint inhibitor targets GITR (eg, anti-GITR antibody). In certain embodiments, targeting GITR inhibits regulatory T cell activity. In certain embodiments, the benefits of combination therapy with a harboxydiene splicing regulator, ADC, or composition with CTLA4 targeting, OX40 targeting, CD40 targeting, and / or GITR targeting agents (eg, at least one). Effects on symptoms or disease progression risk / rate) are additive. In some embodiments, the benefits of combination therapy with a harboxydiene splicing regulator, ADC, or composition with CTLA4 targeting, OX40 targeting, CD40 targeting, and / or GITR targeting agents are hyperadditive. (That is, synergistic).

Checkpoint inhibitor therapeutic strategies are such that treatment promotes and / or enhances priming of T cell responses to tumors with weak or low antigenicity (eg, CTLA4), or responds to tumor antigens, but presents antigen. Based on the hypothesis that T cells that have become "depleted" due to their chronic nature are recovered and / or reactivated by treatment (eg, PD1, PDL1) (Chen and Mellman (2013) Immunoty 39 (E.g., PD1, PDL1). 1): 1-10). Examples of suitable checkpoint inhibitory therapies and agents, such as anti-PD1, anti-PDL1, or anti-CTLA4 antibodies, are known in the art. See, for example, Pamphlet 2001/014424, Pamphlet International Publication No. 2013/173223, Pamphlet International Publication No. 2016/007235.

The combination of these primed T cell responses after checkpoint inhibitor therapy with therapies that induce tumor cells with neoantigens to which the primed immune system can respond can provide beneficial synergies. Concomitant use with CTLA4 inhibitors may be particularly beneficial as harboxydiene splicing regulators or neoantigens resulting from ADCs have not yet been presented for T cell priming. In some embodiments, treatment administers one or more harboxydiene splicing regulators, ADCs, or compositions to induce the production of neoantigen, prior to, simultaneously with, or thereafter, CTLA4 inhibition. The initial administration of the drug is followed by stimulating CD8 T cell priming. In some embodiments, additional administration of a CTLA4 inhibitor is provided to the patient, for example to further stimulate priming and / or activation of a neoantigen-responsive CD8 population. In some embodiments, additional administration of a harboxydiene splicing regulator, ADC, or composition can be given to the patient to increase neoantigen presentation by the tumor. Repeated doses of herboxydiene splicing regulators, ADCs, or compositions and checkpoint inhibitor therapies can be given simultaneously or at staggered intervals. In some embodiments, the treatment is to administer PD1 / PDL1 inhibitor co-treatment, for example thereby further restoring the effector function of depleted neoantigen-targeted T cells in the tumor microenvironment. include.

The term "combination" or "combination therapy", as used herein, is an additional drug or therapy (eg, a checkpoint inhibitor, for example, one or more herboxidienspiring regulators, ADCs, or compositions. To provide a beneficial (ie, additive or synergistic) effect by the interaction of one or more of the drugs administered, together with a cytokine or cytokine analog, neo-antigen vaccine, CAR-T). Refers to administration as part of the intended treatment regimen. In some embodiments, the combination also includes, but is not limited to, a chemotherapeutic agent, an anti-angiogenic agent, and an agent that reduces immunosuppression (eg, a second checkpoint inhibitor). Additional medications may also be included. Beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic interactions resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination is typically carried out over a defined period of time (eg, minutes, hours, days, or weeks depending on the combination of choices).

"Combinated" or "co-administered", when used herein, is two or more different to a subject while the subject is suffering from a medical condition (eg, neoplastic disorder). It means that the treatment is delivered. For example, in some embodiments, two or more treatments are performed after a subject has been diagnosed with a disease or disorder and before the disease or disorder has healed or disappeared, or the subject is at risk for the disease. Delivered when identified, but before the subject develops the condition. In some embodiments, delivery of one treatment is still performed at the start of delivery of the second treatment, and thus there is overlap. In some embodiments, the first and second treatments are initiated simultaneously. This type of delivery is sometimes referred to herein as "simultaneous," "parallel," or "combined" delivery. In another embodiment, delivery of one treatment ends prior to the initiation of delivery of the second treatment. This type of delivery is sometimes referred to herein as "continuous" or "sequential" delivery.

In some embodiments, the two therapies (eg, a harboxydiene splicing regulator, ADC, or composition and checkpoint inhibitor) are included in the same composition. Such compositions may be administered in any suitable form and by any suitable route. In other embodiments, the two therapies (eg, harboxydiene splicing regulators, ADCs, or compositions and checkpoint inhibitors) are separate compositions, in any suitable form, and in any suitable manner. Administered by route. For example, in some embodiments, a composition comprising a harboxydiene splicing regulator or ADC and a composition comprising a checkpoint inhibitor are administered in parallel or sequentially at different times in any order. In either case, they must be administered in close enough time to provide the desired therapeutic or prophylactic effect.

In either embodiment of co-delivery or sequential delivery, the combination administration may make the treatment more effective. In some embodiments, the first treatment is more effective than would have been seen if the first treatment had been administered in the absence of the second treatment, eg, less first. Equivalent effects are seen with the treatment of (eg, at lower doses). In some embodiments, the first treatment is such that reduction of symptoms, or other parameters associated with the disease or disorder, is observed delivering the first treatment in the absence of the second treatment. It is more effective than wax. In other embodiments, a similar situation is observed with the second treatment. In some embodiments, the benefits of combination therapy (eg, the effect on at least one sign or disease progression risk / rate) are additive. In some embodiments, the benefits of combination therapy are hyperadditive.

In various embodiments, the present disclosure relates to a subject having or suspected of having a neoplastic disorder, a composition comprising an effective amount of a harboxydienpiding regulator, an ADC, or a harboxydienspiring regulator or an ADC; Provided is a method of treating by administering to a subject at least one additional therapy (eg, checkpoint inhibitor therapy, cytokine or cytokine analog, neoantigen vaccine, CAR-T). In some embodiments, administration of a harboxydiene splicing regulator, ADC, or composition induces at least one neoantigen and / or T cell response. In some embodiments, administration of a harboxydiene splicing regulator, ADC, or composition induces a double-stranded RNA immune response. In some embodiments, administration of a harboxydiene splicing regulator, ADC, or composition induces immunogenic cell death. In some embodiments, the at least one additional therapy may include at least one, at least two, at least three, at least four, or at least five additional therapies. For example, in some embodiments, the harboxydiene splicing regulator, ADC, or composition may be administered in combination with two checkpoint therapies, i.e., using two different checkpoint inhibitors. good. In some other embodiments, the harboxydiene splicing regulator, ADC, or composition may be administered in combination with checkpoint inhibitor therapy and neoantigen vaccine.

In some embodiments of the combination therapy, the dose of the harboxydien Splicing regulator, antibody-drug conjugate, or composition and / or at least one additional therapy is the harboxydiensplicing regulator, antibody-drug conjugate. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% when compared to the standard dosage of gate or composition and / or at least one additional therapy. , 75%, or 90% reduction. In some embodiments, the harboxydien Splicing regulator, antibody-drug conjugate, or composition and / or at least one additional therapy is a harboxydiensplicing regulator, antibody-drug conjugate, or composition and / Or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or when compared to the standard dosing regimen of at least one additional therapy. It is administered 90% less frequently. In some embodiments, the dose and / or dosage of a harboxydiene splicing regulator, antibody-drug conjugate, or composition and / or at least one additional therapy reduces systemic toxicity and / or is tolerable. Brings improvement in sex.

In some embodiments, administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is initiated prior to administration of at least one additional therapy. In some embodiments, administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition is initiated after administration of at least one additional therapy. In some embodiments, administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is initiated at the same time as administration of at least one additional therapy.

In some embodiments, administration of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is repeated at least once after the initial dose. In some embodiments, the amount used for repeated doses of the harboxydiene splicing regulator, antibody-drug conjugate, or composition is reduced when compared to the amount used for the initial dose. In some embodiments, the amount used for repeated administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition is a harboxydiene splicing regulator, antibody-drug conjugate, or composition. Reduced when compared to standard dosages. In some embodiments, the amount used for repeated administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition is a harboxydiene splicing regulator, antibody-drug conjugate, or composition. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% reduction when compared to standard dosages.

In some embodiments, administration of at least one additional therapy is repeated at least once after the initial administration. In some embodiments, the amount used for repeated doses of at least one additional therapy is reduced when compared to the amount used for the initial dose. In some embodiments, the amount used for repeated doses of at least one additional therapy is reduced when compared to the standard dosage of at least one additional therapy. In some embodiments, the amount used for repeated doses of at least one additional therapy is 10%, 15%, 20%, 25%, when compared to the standard dosage of at least one additional therapy. It is reduced by 30%, 35%, 40%, 45%, 50%, 75%, or 90%.

In some embodiments, repeated doses of harboxydiene splicing regulators, antibody-drug conjugates, or compositions are simultaneous with repeated doses of at least one additional therapy. In some embodiments, repeated doses of harboxydiene splicing regulators, antibody-drug conjugates, or compositions are sequential or staggered with repeated doses of at least one additional therapy.

In various embodiments, the present disclosure comprises a subject having or suspected of having a neoplastic disorder in an effective amount of a harboxydiene splicing regulator, ADC, or harboxydiene splicing regulator or ADC; Provided is a method of treating by administering a checkpoint inhibitor therapy to a subject. In some embodiments, checkpoint inhibitor therapy comprises administering at least one checkpoint inhibitor. In some embodiments, the subject is intolerant, intolerant, or refractory to at least one checkpoint inhibitor when administered alone. In some embodiments, the subject is determined using, for example, an immune-related Response Criteria (irRC) and / or an immune-related solid cancer effect criteria (Response Assessment Criteria in Solid Tumors: irRECIST). When so, it can be considered refractory or refractory to at least one checkpoint inhibitor. For example, Wolchok et al. (2009) Clin Cancer Res. 15 (23): 7412-20; Bonsack et al. See "Adaptation of the Immuno-Returned Response Criteria: irRECIST" (Abstract 4958) ESMO 2014. Illustrative criteria remain the same when the treatment being evaluated is an immuno-oncological drug (eg, a checkpoint inhibitor) and the tumor of the cancer patient improves (“responds”) during treatment. It may include those used in the art to define when (“stabilizes”) or worsens (“progresses”). In some embodiments, if the subject has one or more adverse (grade 2 or higher) events identified for each checkpoint inhibitor (eg, ipilimumab), the subject is at least one. One checkpoint inhibitor can be considered intolerant. In some embodiments, if the subject has one or more adverse events selected from, for example, enteritis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and endocrine disease, the subject is It may be considered intolerant to the treatment of ipilimumab (Yervoy® (ipilimumab) FDA Labeling Supplement, 2018).

In some embodiments, the checkpoint inhibitor targets PD1 / PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and / or KIR. In some embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR. In some embodiments, the checkpoint inhibitor is targeted with an inhibitory antibody or other similar inhibitory molecule. In some other embodiments, the checkpoint inhibitor is targeted with an agonist antibody or other similar agonist molecule. In some embodiments, the checkpoint inhibitor comprises a cytotoxic T lymphocyte-related antigen 4 pathway (CTLA4) inhibitor. In some embodiments, the CTLA4 inhibitor is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody is ipilimumab. In some embodiments, the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor. In some embodiments, the PD1 inhibitor is an anti-PD1 antibody. In some embodiments, the anti-PD1 antibody is nivolumab. In some embodiments, the PD1 inhibitor is an anti-PDL1 antibody. In some embodiments, the anti-PDL1 antibody is atezolizumab. In some embodiments, checkpoint inhibitors include CTLA4 inhibitors and PD1 inhibitors. In some embodiments, the checkpoint inhibitor targets OX40. In some embodiments, the checkpoint inhibitor targets CD40. In some embodiments, the checkpoint inhibitor targets the GITR. In some embodiments, harboxydienspiring regulators, ADCs, or compositions and checkpoint inhibitors (eg, CTLA4 targeting, PD1 / PDL1 targeting, OX40 targeting, CD40 targeting, and / or GITR targeting. The benefits of combination therapy with (eg, antibodies or molecules) (eg, the effect on at least one symptom or disease progression risk / rate) are additive. In some embodiments, harboxydiene splicing regulators, ADCs, or compositions and checkpoint inhibitors (eg, CTLA4 targeting, PD1 / PDL1, OX40 targeting, CD40 targeting, and / or GITR targeting antibodies. The benefits of combination therapy with (or molecules) are hyperadditive (ie, synergistic).

In various embodiments, the present disclosure comprises a subject having or suspected of having a neoplastic disorder in an effective amount of a harboxydiene splicing regulator, ADC, or harboxydiene splicing regulator or ADC; Provided is a method of treating by administering a cytokine or a cytokine analog therapy to a subject. In some embodiments, cytokine or cytokine analog therapy comprises administering at least one cytokine or cytokine analog. In some embodiments, the subject is intolerant, intolerant, or refractory to at least one cytokine or cytokine analog when administered alone.

In some embodiments, the cytokine or cytokine analog comprises a T cell enhancer. In some embodiments, cytokines or cytokine analogs include IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα. In some embodiments, cytokines or cytokine analogs include IL-2, IL-10, IL-12, and / or IL-15. In some embodiments, administration of a cytokine or cytokine analog results in T cell priming after administration of a harboxydiene splicing regulator, antibody-drug conjugate, or composition due to the induction and presentation of neoantigen. Is enhanced.

In some embodiments, the cytokine or cytokine analog comprises IL-2. In some embodiments, IL-2 boosts the signal to effector cells and promotes their expansion (Rosenberg (2014) J Immunol. 192 (12): 5451-8). In some embodiments, the cytokine or cytokine analog comprises IL-10. In some embodiments, IL-10 boosts CD8 + T cell priming and activation (Mumm et al. (2011) Cancer Cell 20 (6): 781-96). In some embodiments, the cytokine or cytokine analog comprises IL-12. In some embodiments, IL-12 links the innate and adaptive immune responses to boost antigen-specific priming and targeting (Tugues et al. (2015) Cell Death Differ. 22 (2): 237-. 46). In some embodiments, the cytokine or cytokine analog comprises IL-15. In some embodiments, IL-15 boosts T effector (CD8) cell priming and / or activation. In some embodiments, the cytokine or cytokine analog comprises IFNγ. In some embodiments, IFNγ captures IFNγ secretion by T effector cells. In some embodiments, the cytokine or cytokine analog comprises TNFα. In some embodiments, TNFα supplements TNFα secretion by T effector cells.

In some embodiments, the initial dose of a harboxydiene splicing regulator, ADC, or composition is administered to the subject to induce aberrant splicing and neoantigen peptide production. After sufficient time for protein production and antigen presentation, in some embodiments, the subject is then administered an initial dose of cytokine or cytokine analog to boost effector T cell priming and expansion, and / or. Promote. In some embodiments, the waiting period between doses of a harboxydiene splicing regulator, ADC, or composition with a cytokine or cytokine analog is about 2, about 3, about 4, about 5, about 6, or It's about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the cytokine or cytokine analog is IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα. In some embodiments, the benefit of a combination therapy of a harboxydiene splicing regulator, ADC, or composition with a cytokine or cytokine analog can be additive or hyperadditive.

In some other embodiments, initial doses of cytokines or cytokine analogs are administered to the subject to boost and / or enhance effector T cell priming and expansion. After the waiting period, in some embodiments, the subject is then administered an initial dose of a harboxydiene splicing regulator, ADC, or composition to induce aberrant splicing and neoantigen peptide production. In some embodiments, the waiting period between doses of a cytokine or cytokine analog and a harboxydiene splicing regulator, ADC, or composition is about 2, about 3, about 4, about 5, about 6, or It's about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the cytokine or cytokine analog is IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα. In some embodiments, the benefit of combination therapy with a cytokine or cytokine analog and a harboxydiene splicing regulator, ADC, or composition can be additive or hyperadditive.

In some embodiments, after a period sufficient for T cell priming and expansion, the subject is then administered a second or subsequent dose of a harboxydiene splicing modulator, ADC, or composition. Induces representation of antigenic peptides. In some embodiments, the waiting period between the initial dose of the cytokine or cytokine analog and the second or subsequent dose of the harboxydiene splicing regulator, ADC, or composition is about 2, about 3, About 4 or about 5 weeks. In some embodiments, the waiting period is about 3 weeks. In some embodiments, subsequent doses of cytokines or cytokine analogs may be administered, eg, interrupted, between subsequent doses of a harboxydiene splicing regulator, ADC, or composition. Upon receiving a second or subsequent dose of a harboxidien splicing regulator, ADC, or composition, in some embodiments, the immune system associates with neoantigen-presenting tumor cells and / or causes tumor cell killing. Can trigger. In some embodiments, after this exemplary initial treatment regimen, administration of a harboxydiene splicing regulator, ADC, or composition can be pulsed, ie, a harboxydiensplicing regulator, ADC, or. The composition is at long enough intervals for antigen presentation, T cell association and / or tumor cell killing, and / or memory T cell population recovery (eg, about every 4 weeks, about every 5 weeks, about every 6 weeks). ) May be administered.

In some embodiments, the subject has a non-synonymous mutation load of about 150 or less mutations. In some embodiments, the subject has a non-synonymous mutation load of about 100 or less mutations. In some embodiments, the subject has a non-synonymous mutation load of about 50 or less mutations. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematological malignancies or solid tumors. In some embodiments, the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. In some embodiments, the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, solid tumors include breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary tract cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and Selected from esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

Harboxydiene splicing regulator / ADC in combination with neoantigen vaccine:
In various embodiments, patients with cancer as described herein can be treated with a harboxydiene splicing regulator, ADC, or composition in combination with a neoantigen vaccine. Although not bound by theory, vaccines used alone or in combination with immune checkpoint inhibitor (ICI) molecules have been promising in early trials (Ott et al. (2017) Nature. 547 (7662): 217-21; Sahin et al. (2017) Nature 547 (7662): 222-6), generally requiring sequencing of patient tumor mutations (Ott et al. (2017) Nature 547 (Ott et al. (2017) Nature 547). 7662): 217-21; Aldous and Dong (2018) Bioorg. Med. Chem. 26 (10): 2842-9). Thus, vaccines often depend on a sufficient number of antigenic non-synonymous mutations. In general, tumors with extremely low mutation loading provide few antigen candidates, and rapidly growing tumors have limited time available for identification and production of patient-specific vaccines.

To date, attempts to develop vaccines that may have broad immunogenicity in most patients have either been mutated frequently, ectopically overexpressed, or amplified. The focus is on such proteins and / or proteins that exist as "self" proteins in the organism. In addition, these proteins are often expressed in immunologically limited tissues (eg, neuronal markers expressed in neuroendocrine tumor types), while others are normal during embryogenesis. Can be expressed (eg, carcinoembryonic antigen). Therefore, the usefulness of vaccines using such proteins as antigens is often limited to the particular tumor lineage or subset in which one or more of the antigens is presented. The usefulness of the vaccine may also need to be confirmed by sequencing patient tumor samples, which can be time consuming.

Furthermore, if these antigens are present as "self" proteins, the immune system may be primed to recognize them as "self" and thus may not respond. Alternatively, if the immune system is capable of initiating an effector response to such antigen, it can lead to on-target side effects in tissues in which the antigen can be expressed. In any of these cases, one of the key challenges is that many antigenic peptides are mutated or amplified in the "passenger" gene (ie, during tumor development, but the tumor itself is alive or alive or amplified. It is derived from a gene that does not play a decisive role in continuing proliferation). Thus, these genes can be silenced without significant impact on tumor progression, thus allowing the tumor to "escape" the immune response to their antigens. Although not desired to be constrained by theory, this mechanism can play a role in tumor evolution, where strong antigenic random mutations are often in the early stages of tumor development. "Counter-selection" by (Dunn et al. (2004) Annu. Rev. Immunol. 22: 329-60).

In addition, some evidence has shown that chronic antigen presentation and immune stimulation can lead to immune cell anergy and depletion (Pardol (2012) Nat. Rev. Cancer 12 (4): 252-64). As shown, ICI either suppresses the depleted immune cell phenotype (α-PD1 / PD-L1) or promotes further immune cell responses (α-CTLA4). These phenotypes form the basis of the rationale behind the current ICI treatment. Notably, with α-CTLA4 therapy, some patients have serious immune-related adverse effects that can be attributed to disruption of immune tolerance mechanisms that promote T cell activation and suppress autoreactive immune responses. It has been reported to present an event.

Both of these techniques (ie, inducing or enhancing the de novo immune response to neoantigens or desuppressing the anergy or depletion of existing immune responses) are associated with chronic immune activation. Thus, these techniques are sensitive to anergy, editing, and other tumor-mediated mechanisms designed to suppress immune associations.

In contrast, treatment with the harboxydiene splicing regulators, ADCs, or compositions disclosed herein can elicit an immune response against the sequence corresponding to the neoantigen. In some embodiments, presentation of neoantigens further diversifies the targets that the adaptive immune system associates and activates. In some embodiments, the harboxydiensplicing regulator, ADC, or composition is chronic to mutation-derived neoantigens by being able to acutely induce alternative splicing and the resulting neoantigen. The risk of immune system fatigue due to exposure may be reduced and / or the ability of tumor cells to adapt to avoid therapy may be limited. In some embodiments, administration of a harboxydiene splicing regulator, ADC, or composition in combination with a neoantigen vaccine immunizes against the neoantigen produced by the harboxydiene splicing regulator, ADC, or composition. The response is enhanced. In some embodiments, the harboxydiene splicing regulator, ADC, or composition is administered before, during, or after vaccination. In some embodiments, the harboxydiene splicing regulator, ADC, or composition and / or vaccine may be administered once or more than once during the course of treatment. In some embodiments, the vaccine is administered once during the course of treatment and the harboxydiene splicing regulator, ADC, or composition is administered more than once. In some embodiments, the vaccine is administered once during the course of treatment, followed by one or more boosters.

As used herein, the term "neoantigen vaccine" refers to one or more immunogenic neoantigen peptides or mRNAs, such as at least 2, at least 3, at least 4, at least 5 or more neos. Refers to a pool sample of antigenic peptides. The term "vaccine" refers to a composition that gives rise to immunity for the prevention and / or treatment of a disease (eg, neoplastic disorder, eg, hematological malignancies or solid tumors). Thus, vaccines are pharmaceuticals containing immunogenic agents and are intended to be used to produce specific immune defenses and protective substances after vaccination in humans or animals. Neoantigen vaccines can further include pharmaceutically acceptable carriers, diluents, excipients, and / or adjuvants.

As used herein, the term "immunogenicity" refers to any agent or composition capable of eliciting an immune response, eg, a T cell response. The immune response can be antibody-mediated, cell-mediated, or both.

In some embodiments, the patient is given a harboxydienplising regulator, ADC, or composition, followed by a known neoantigen peptide or mRNA vaccine, thereby giving the harboxydiensplicing regulator, ADC, or composition. Enhances the immune response to the neoantigen produced by the composition. In some other embodiments, the patient is given a harboxydiene splicing regulator, ADC, or composition and screened for the neoantigen produced by the treatment. One or more of those neoantigens are then used to create a personalized vaccine to be given to the patient. In any of these embodiments, the harboxydiene splicing regulator, ADC, or composition and / or peptide or mRNA vaccine may be administered to the patient once or repeatedly.

In various embodiments, a suitable neoantigen for a vaccine is to screen a panel of transcripts with altered splicing (eg, from a tumor biopsy) and robust expression from one or more tissue samples of a patient. Can be identified by. In some embodiments, the mutant protein sequence spans an abnormally spliced mRNA junction in the screened sample, retaining a portion of the protein sequence (up to 12 amino acids) flanking amino acid changes across the junction. Identified based on translation. In some embodiments, peptide fragments across these junctions are scanned for high affinity binding to the MHC1 allele using tools such as NetMHC1 (Nielsen et al. (2003) Protein Sci 12 (5)). : 1007-17; Andreatta and Nielsen (2016) Bioinformatics 32 (4): 511-7). These results filter the neopeptides to be the expected high affinity binders for the unique patient HLA allele composition, as well as the expected widespread binding to the HLA alleles that are frequently found in different populations. It is possible to assemble a pool of peptides (Maiers et al. (2007) Hum Immunol 68 (9): 779-88). In various embodiments, the identified neopeptide is then formulated as a vaccine, eg, by conjugation with a suitable carrier or adjuvant (Ott et al. (2017) Nature 547 (7662): 217-21. ), Or formulated for delivery as mRNA (Sahin et al. (2017) Nature 547 (7662): 222-6).

In some embodiments, the neoantigen selected is one or more neoantigens resulting from treatment by screening the tumor response of an individual patient (patent) to a harboxydienspiring regulator, ADC, or composition. Based on identification for use in subsequent vaccinations. In other embodiments, neoantigens are selected based on identifying a common neoantigen produced by a harboxydiene splicing regulator, ADC, or composition, eg, by screening sample panels from different patients. , Then used as a universal vaccine for future patients.

Although not constrained by theory, in some embodiments, with the use of universal neoantigen vaccines, the neoantigen selected is not tumor mutation dependent, but rather a harboxidien splicing regulator, ADC, Alternatively, to mimic the neoantigen produced by the composition and generally recognized as exogenous by the organism, the need for sequencing and analysis of the unique mutational state of each patient's tumor can be avoided. In addition, in some embodiments, the patient's tumor cells are dependent on tumor mutations when compared to those that mimic the neoantigen produced by the harboxidien splicing regulator, ADC, or composition1. The use of neoantigen vaccines can be particularly effective because they can be more likely to mutate away from the production of one or more neoantigens. This allows the formulation of bulk vaccines that can be broadly immunogenic to most patients and may accelerate the initiation of treatment regimes. Patients can be vaccinated according to the schedule outlined herein and prior to the completion of subsequent vaccination, for example to induce expression of neoantigen peptides, such as harboxidien splicing regulators, ADCs, or compositions. May be further treated by. In some embodiments, the patient may administer a harboxydiene splicing regulator, ADC, or composition prior to, simultaneously with, or after vaccination. In some embodiments, the patient is administered a harboxydiene splicing regulator, ADC, or composition and is screened for one or more neoantigens found in a panel of universal neoantigens, at least identified in the subject. Inoculated with a universal neoantigen vaccine containing one universal neoantigen. In some embodiments, the patient may receive the harboxydiene splicing regulator, ADC, or composition once or more than once after vaccination. The harboxydiene splicing regulator or ADC or composition and / or vaccine may be administered once or more than once during the course of treatment.

In various embodiments, the vaccine may comprise one or more neoantigen peptides or mRNAs. In various embodiments, the vaccine may comprise one or more long neoantigen peptides. Such "long" neoantigen peptides, in various embodiments, cause efficient internalization, processing, and cross-presentation in professional antigen presenting cells such as dendritic cells. Similarly, long vaccine peptides have been shown to induce cytotoxic T cells in humans in other contexts (Melief and van der Burg (2008) Nat Rev Cancer 8 (5): 351-60). .. In various embodiments, the neoantigen peptide is extended to include the neoantigen peptide sequence itself in addition to the adjacent amino acid sequence. In various embodiments, the extended peptide sequence facilitates protein uptake by antigen presenting cells, such as dendritic cells. In various embodiments, the extended peptide sequence allows efficient antigen presentation and T cell priming in models of different HLA isotypes. In various embodiments, long neoantigen peptides and / or extended peptide sequences are compared to short neoantigen peptides and / or short peptide sequences (eg, peptide sequences less than about 10 amino acids long or less than about 5 amino acids long). , Increased uptake by antigen presenting cells (eg, dendritic cells), increased antigen presentation, and / or increased T cell priming. In some embodiments, the long neoantigen peptide ranges from about 5 to about 50 amino acids in length. In some embodiments, the long neoantigen peptide ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the long neoantigen peptide ranges from about 15 to about 25 amino acids in length.

In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety does not exclusively overlap or overlap with the canonical peptide sequence (eg, any of the exemplary canonical peptide sequences underlined in Table 8). Then it doesn't.

The amino acid sequences of the exemplary long neoantigen peptides are shown in Table 8.

These exemplary neoantigen peptides are produced after administration of an ADC containing a pragienolide splicing regulator, however, given a similar mechanism of action (ie, a similar splicing regulatory mechanism). , Harboxydiene splicing regulators can produce similar neoantigen peptides.

The protein sequences of the 29 neopeptides listed in Table 7 can be extended. The extended protein sequence incorporates both the neopeptide sequence itself in addition to the adjacent amino acid sequence. Elongated protein sequences better promote protein uptake by dendritic cells, allowing antigen presentation and T cell priming in models of different HLA isotypes. The amino acid sequences of the 29 extended neopeptides are shown in Table 8.

As used herein, a neoantigen peptide or mRNA vaccine comprises the use of a fragment of a neoantigen peptide or a coding mRNA thereof, as long as the fragment retains its immunogenic potential.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide. In some embodiments, the neoantigen vaccine is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 neo. Contains antigenic peptides. In some embodiments, the one or more neoantigen peptides range from about 5 to about 50 amino acids in length. In some embodiments, the one or more neoantigen peptides range from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the one or more neoantigen peptides range from about 15 to about 25 amino acids in length.

In various embodiments, the present disclosure comprises a subject having or suspected of having a neoplastic disorder in an effective amount of a harboxydiene splicing regulator, ADC, or harboxydiene splicing regulator or ADC; and Provided is a method of treating a subject by administering a neoantigen vaccine. The neo-antigen vaccine may be, for example, a peptide or mRNA neo-antigen vaccine. In some embodiments, the harboxydiene splicing regulator, ADC, or composition is administered prior to administration of the neoantigen vaccine. In some embodiments, the harboxydiene splicing regulator, ADC, or composition is administered after administration of the neoantigen vaccine. In some embodiments, the harboxydiene splicing regulator, ADC, or composition is administered at the same time as the administration of the neoantigen vaccine. In some embodiments, administration of the harboxydiene splicing regulator, ADC, or composition is repeated at least once after the initial dose. In some embodiments, the amount used for repeated doses of the harboxydiene splicing regulator, ADC, or composition is reduced when compared to the amount used for the initial dose.

In various embodiments, the present disclosure comprises a harboxidiene splicing regulator, an ADC, or a harboxidiene splicing regulator or ADC for use in the treatment of a subject having or suspected of having a neoplastic disorder. The combination with a neo-antigen vaccine (eg, a universal neo-antigen vaccine) is further provided. In some embodiments, the neoantigen vaccine is a peptide or mRNA neoantigen vaccine. In some embodiments, the combination further comprises at least one additional therapy. In some embodiments, the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

In various embodiments, the present disclosure comprises a subject having or suspected of having a neoplastic disorder, (a) an effective amount of a harboxidi-splicing regulator, an ADC, or a harboxydi-splicing regulator or an ADC. Administering a substance to a subject; (b) detecting one or more neoantigens in a subject after administration of a harboxydienspiring regulator, ADC, or composition; (c) universalizing one or more neoantigens. Comparing with a panel of neoantigens; and (d) further provide a method of treating by administering to a subject a universal neoantigen vaccine comprising at least one universal neoantigen present in the subject. In some embodiments, the universal neoantigen vaccine is administered alone or in combination with at least one additional therapy. In some embodiments, the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

In some embodiments, at least one additional therapy comprises repeated administration of a harboxydiene splicing regulator, ADC, or composition. In some embodiments, repeated administration of the harboxydiene splicing regulator, ADC, or composition is initiated prior to administration of the universal neoantigen vaccine. In some embodiments, repetition of the harboxydiene splicing regulator, ADC, or composition is initiated after administration of the universal neoantigen vaccine. In some embodiments, repeated administration of the harboxydiene splicing regulator, ADC, or composition is initiated at the same time as administration of the universal neoantigen vaccine. In some embodiments, the amount used for repeated doses of the harboxydiene splicing regulator, ADC, or composition is reduced when compared to the amount used for the initial dose. In some embodiments, the amount used for the initial and / or repeated doses of the harboxydiene splicing regulator, ADC, or composition is the harboxydiene splicing regulator when used without vaccine treatment. It is reduced when compared to the standard dosage of ADC, or composition. In some embodiments, the amount used for the initial and / or repeated doses of the harboxydiene splicing regulator, ADC, or composition is standard for the harboxydiene splicing regulator, ADC, or composition. When compared to the dosage, it is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%.

In some embodiments, at least one additional therapy comprises administering a checkpoint inhibitor (eg, any of the exemplary checkpoint inhibitors described herein). In some embodiments, administration of the checkpoint inhibitor is initiated prior to administration of the universal neoantigen vaccine and / or repeated administration of the harboxydiene splicing regulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is initiated after administration of the universal neoantigen vaccine and / or repetition of the harboxydiene splicing regulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is initiated at the same time as administration of the universal neoantigen vaccine and / or repeated administration of the harboxydiene splicing regulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is repeated at least once after the initial administration. In some embodiments, the amount used for repeated doses of the checkpoint inhibitor is reduced when compared to the amount used for the initial dose. In some embodiments, the amount used for repeated doses of the checkpoint inhibitor is reduced when compared to the standard dosage of the checkpoint inhibitor. In some embodiments, the amount used for repeated doses of the checkpoint inhibitor is 10%, 15%, 20%, 25%, 30% when compared to the standard dosage of the checkpoint inhibitor. , 35%, 40%, 45%, 50%, 75%, or 90% reduction. In some embodiments, the subject is intolerant, intolerant, or refractory to the checkpoint inhibitor when administered alone.

Also provided herein are neoantigen vaccines comprising at least one neoantigen peptide or at least one neoantigen mRNA in various embodiments. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide. In some other embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA.

Also described herein are compositions comprising a harboxydiene splicing regulator, an ADC, or a harboxydiene splicing regulator or ADC in various embodiments; a neoantigen vaccine (eg, a universal neoantigen vaccine). A kit including is also provided. In some embodiments, the neoantigen vaccine is a peptide or mRNA neoantigen vaccine. In some embodiments, the kit is not limited to instructions for use; other agents, such as one or more additional therapeutic agents; harboxidien splicing regulators, ADCs, compositions, and / or neoantigens. Devices, containers, or other materials for preparing vaccines for therapeutic administration; pharmaceutically acceptable carriers; and harboxidien splicing regulators, ADCs, compositions, and / or neoantigen vaccines for patients. It further comprises one or more additional components, including a device, container, or other material for administration. Instructions for use may include guidance on therapeutic applications, including recommended dosages and / or methods of administration, for example in patients with or suspected of having neoplastic disorders. In various embodiments, the kit further relates to therapeutic use, eg, the use of a harboxydiene splicing regulator, ADC, or composition for treating or preventing a patient's neoplastic disorder, and the use of a neoantigen vaccine. Contains instructions. In various embodiments, the kit is further for administration with at least one additional therapeutic agent (eg, a harboxydiene splicing regulator, ADC, or composition, and neoantigen vaccine, eg, check). (Point inhibitor) is included. In various embodiments, the harboxydiene splicing regulator, ADC, composition, and / or neoantigen vaccine is formulated as a pharmaceutical composition.

In some embodiments of the methods and compositions disclosed herein, a neoantigen vaccine comprises at least one neoantigen peptide. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length.

In some embodiments, the at least one neoantigen peptide comprises one or more of the neoantigen sequences disclosed herein.

In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety does not exclusively overlap or overlap with the canonical peptide sequence (eg, any of the exemplary canonical peptide sequences underlined in Table 8). Then it doesn't.

In some embodiments, the neoantigen sequence is a subject-specific neoantigen sequence. In some embodiments, the neoantigen sequence is a neoantigen vaccine personalized for the subject. In some embodiments, the neoantigen sequence has the ability to bind to at least one HLA allele expressed in the subject.

In some other embodiments, the neoantigen sequence is a universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal neoantigen vaccine. In some embodiments, the neoantigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% of subjects in a population of subjects suffering from neoplastic disorders. It has the ability to bind to at least one HLA allele that is expressed in at least 40%, or at least 45%. In some embodiments, the neoantigen sequence has the ability to elicit a T cell response to tumors present in at least 1%, at least 5%, or at least 10% of the subject population suffering from neoplastic disorders.

In some embodiments, the neoantigen sequence sequences at least one neoantigen peptide induced in the subject by administering an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition. Have been identified by doing. In some embodiments, the at least one neoantigen peptide comprises a neoantigen sequence derived by contacting an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition with neoplastic cells. .. In some embodiments, neoplastic cells are present in in vitro cell cultures. In some embodiments, neoplastic cells are obtained from the subject. In some embodiments, neoplastic cells are present in the subject.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide or mRNA and a pharmaceutically acceptable carrier. In various embodiments, the neoantigen peptide or mRNA can be linked to a suitable carrier that helps elicit an immune response. Exemplary carriers for linking to immunogenic agents (eg, neoantigenic peptides or mRNAs) include serum albumin, keyhole limpet hemocianine, immunoglobulin molecules, tyroglobulin, ovalbumin, tetanus toxoid, or other pathogens. From sex bacteria, such as diphtheria, E. coli, cholera, or H. Examples include toxoids such as H. pylori, or attenuated toxin derivatives. Other carriers for stimulating or enhancing the immune response include cytokines such as IL-1, IL-1α and β peptides, IL-2, γINF, IL-10, GM-CSF, and chemokines such as chemokines. Examples thereof include M1P1α and β and RANTES. Immunogenic agents can also be linked to peptides that enhance intertissue transport, as described, for example, in WO 97/17613 and Pamphlet 97/17614. In some embodiments, the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. Toxoid.

In some embodiments, the neoantigen peptide or mRNA may be linked to a pharmaceutically acceptable carrier. The immunogenic agent can be linked to the carrier by chemical cross-linking. Techniques for linking immunogenic peptides to carriers include N-succinimidyl-3- (2-pyridyl-thio) propionate (SPDP) and succinimidyl 4- (N-maleimidemethyl) cyclohexane-1-carboxylate (SMCC) (SMCC). If the peptide lacks a sulfhydryl group, this may be provided by the addition of a cysteine residue) to include the formation of disulfide bonds. These reagents create disulfide bonds between themselves and peptide cysteine residues on one protein and create amide bonds via ε-amino on lysine or other free amino groups on other amino acids. .. Various such disulfide / amide formers are described in Jansen et al. ((1982) Immuno Rev. 62: 185). Other bifunctional coupling agents form thioethers rather than disulfide bonds. Many of these thioether forming agents are commercially available, with reactive esters of 6-maleimide caproic acid, 2-bromoacetic acid, and 2-iodoacetic acid, 4- (N-maleimide-methyl) cyclohexane-1-carboxylic acid. Can be mentioned. The carboxyl group can be activated by combining it with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salts. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are covalently linked via a linker.

Neoantigens and other such immunogenic peptides can also be expressed as fusion proteins with carriers. The immunogenic peptide can be linked (internally) to the carrier at any site within the amino-terminal, carboxyl-terminal, or peptide. In some embodiments, the fusion protein may have multiple repeats of the immunogenic peptide. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as fusion proteins.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide or coding mRNA thereof and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide or a coding mRNA thereof and a pharmaceutically acceptable adjuvant (eg, an adjuvant as described herein).

In some embodiments of the methods and compositions disclosed herein, a neoantigen vaccine comprises at least one neoantigen mRNA. In some embodiments, at least one neoantigen mRNA encodes one or more neoantigen sequences.

In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and / or the antigenic moiety does not exclusively overlap or overlap with the canonical peptide sequence (eg, any of the exemplary canonical peptide sequences underlined in Table 8). Then it doesn't.

In some embodiments, the neoantigen sequence is a subject-specific neoantigen sequence. In some embodiments, the neoantigen sequence is a neoantigen vaccine personalized for the subject. In some embodiments, the neoantigen sequence has the ability to bind to at least one HLA allele expressed in the subject.

In some other embodiments, the neoantigen sequence is a universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal neoantigen vaccine. In some embodiments, the neoantigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% of subjects in a population of subjects suffering from neoplastic disorders. It has the ability to bind to at least one HLA allele that is expressed in at least 40%, or at least 45%. In some embodiments, the neoantigen sequence has the ability to elicit a T cell response to tumors present in at least 1%, at least 5%, or at least 10% of the subject population suffering from neoplastic disorders.

In some embodiments, the neoantigen sequence sequences at least one neoantigen mRNA induced in a subject by administering an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition. Have been identified by doing. In some embodiments, the at least one neoantigen mRNA provides a neoantigen sequence derived by contacting an effective amount of a harboxydiene splicing regulator, antibody-drug conjugate, or composition with neoplastic cells. Code. In some embodiments, neoplastic cells are present in in vitro cell cultures. In some embodiments, neoplastic cells are obtained from the subject. In some embodiments, neoplastic cells are present in the subject.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier. In some embodiments, at least one neoantigen mRNA is ligated to a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. Toxoid.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable adjuvant (eg, an adjuvant as described herein).

In some embodiments, the neoantigen mRNA is encapsulated in an encapsulant. In some embodiments, the encapsulant protects the neoantigen mRNA from degradation and improves vaccine delivery (McNamara et al. (2015) J Immunol Res. 2015: 794528). In some embodiments, the encapsulant is a liposome. In some embodiments, the liposomes are cationic liposomes such as N- [1- (2,3-dioleolioxy) propyl] -N, N, N-trimethylammonium chloride 1 (DOTAP). be. In some embodiments, the encapsulant is nanoparticles. In some embodiments, the nanoparticles protect the neoantigen mRNA from nuclease degradation and / or enhance cell uptake and / or delivery efficiency. In some embodiments, the nanoparticles may be modified to be completely degradable. In some embodiments, the nanoparticles are biodegradable core-shell structure nanoparticles in which the pH responsive poly- (b-aminoester) (PBAE) core is wrapped in a phospholipid shell (Sue et al. (2011) Mol Palm. 8 (3): 774-87). In some embodiments, such nanoparticles are particularly efficient in delivering mRNA in vivo and inducing an antitumor immune response.

In some embodiments, the subject has a non-synonymous mutation load of about 150 or less mutations. In some embodiments, the subject has a non-synonymous mutation load of about 100 or less mutations. In some embodiments, the subject has a non-synonymous mutation load of about 50 or less mutations. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematological malignancies or solid tumors. In some embodiments, the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. In some embodiments, the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, solid tumors include breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary tract cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and Selected from esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

As used herein, "assistant" refers to a substance that has the ability to increase, amplify, or regulate an immune response to an associated immunogenic agent, such as a neoantigenic peptide or mRNA. In certain embodiments, the neoantigens of the present disclosure can be administered in combination with an adjuvant, i.e., a substance that does not elicit an adaptive immune response by itself but amplifies or regulates the response to an accompanying neoantigen. .. Various adjuvants can be used in combination with the disclosed neoantigens to elicit an immune response. In some embodiments, the one or more adjuvants are selected to enhance the unique response to the neoantigen without causing conformational changes in the neoantigen that may affect the qualitative form of the response. .. In some embodiments, the one or more adjuvants are selected to enhance T effector (eg, CD8) cell priming and / or activation.

In certain embodiments, the adjuvant is an aluminum salt (alum) such as aluminum hydroxide, aluminum phosphate, and aluminum sulphate. Such adjuvants may or may not include other specific immunostimulants such as 3-O-deacylated monophosphoryl lipid A (MPL) or 3-DMP, polymers such as polyglutamic acid or polylysine or monomeric amino acids. Can be used. Such adjuvants include muramyl peptides (eg, N-acetylmuramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramil-L-alanine-D-isoglutamine (nor-MDP), N-Acetyl Muramyl-L-alanyl-D-isoglutamine-L-alanine-2- (1'-2'dipalmitoyle-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), N-acetyl Other specifics such as glucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP)) or other bacterial cell wall constituents. Can be used with or without the immunostimulatory agent of. Another adjuvant is an oil-in-water emulsion, 5% squalene, 0.5% Tween 80 formulated into submicron particles using a microfluidizer such as (a) Model 110Y Microfluidics. MF59 (International Publication No. 90/14837) containing 0.5% Span 85 (optionally containing various amounts of MTP-PE), (b) microfluidized into a submicron emulsion. Contains 10% squalene, 0.4% Tween 80, 5% pluronic blocked polymer L121, and thr-MDP, either vortexed to produce larger particle size emulsions. SAF, and (c) 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall constituents, monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS). ), For example, Ribi ™ adjuvant system (RAS) (Ribi ImmunoChem) containing MPL-FCWS (Detox ™). In some embodiments, the adjuvant is a saponin such as Regulon ™ (QS21), or particles produced from it such as ISCOM (Immune Stimulating Complex) and ISCOMARTIX. Other adjuvants include complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF). ), Tumor necrosis factor (TNF) and the like.

The adjuvant can be administered as a single composition with an immunogenic agent (eg, neoantigenic peptide or mRNA), or can be administered before, simultaneously with, or after the administration of the immunogenic agent. In some embodiments, the immunogenic agent and the adjuvant can be packaged and supplied in the same vial, or can be packaged in separate vials and mixed prior to use. In some embodiments, immunogenic agents and adjuvants can be packaged with indications indicating their intended therapeutic application. In some embodiments, if the immunogenic agent and the adjuvant are packaged separately, the packaging may include instructions to mix before use. The choice of adjuvant and / or carrier depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, the effectiveness of the adjuvant for the species to be vaccinated, and is pharmaceutically acceptable in humans. An adjuvant is one that has been approved or is expected to be administered to humans by the relevant regulatory authority. For example, a complete Freund's adjuvant is not suitable for human administration. However, alum, MPL or incomplete Freund's adjuvant (Chang et al. (1998) Adv Drag Deliv Rev. 32: 173-186) may be either alum, QS21, and MPL alone or optionally. , And in combination with all of these, suitable for human administration.

In various embodiments, the present disclosure further provides a method of screening and identifying at least one neoantigen. More specifically, in various embodiments, the present disclosure comprises contacting a neoplastic cell with (a) an effective amount of a harboxydiensplicing regulator, an ADC, or a composition comprising a harboxydiensplicing regulator or an ADC. (B) Detecting at least one selectively spliced mRNA transcript after contacting the neoplasmic cells with a harboxydien splicing regulator, ADC, or composition; (c) at least 1 Predicting translation of one selectively spliced mRNA transcript into at least one peptide; and (d) providing a method of identifying at least one neoantigen by comparing at least one peptide with a reference proteome. And where at least one peptide does not match any peptide of the reference proteome, then at least one neoantigen is identified. In various embodiments, the method further comprises contacting with one or more additional neoplastic cells to identify at least one universal neoantigen. In various embodiments, repeating the process with one or more additional neoplastic cells or samples (eg, tissue biopsy) confirms suitable neoantigens (eg, for use in neoantigen vaccines). And / or one or more universal neoantigens are identified.

In various other embodiments, the present disclosure comprises contacting a neoplastic cell with a composition comprising (a) an effective amount of a harboxydien splicing regulator, an ADC, or a harboxydien splicing regulator or an ADC; b) After contacting the harboxydiensplicing regulator, ADC, or composition with the neoplastic cells, detect at least one peptide containing a potential neoantigen sequence; and (c) see at least one peptide. It provides a method of identifying at least one neoantigen by comparison with a proteome, where at least one neoantigen is identified if at least one peptide does not match any peptide of the reference proteome. In various embodiments, the method further comprises contacting with one or more additional neoplastic cells to identify at least one universal neoantigen. In various embodiments, repeating the process with one or more additional neoplastic cells or samples (eg, tissue biopsy) confirms suitable neoantigens (eg, for use in neoantigen vaccines). And / or one or more universal neoantigens are identified.

In some embodiments of the neoantigen identification methods described herein, detecting at least one selectively spliced mRNA transcript comprises RNAseq. In some embodiments, predicting translation of at least one selectively spliced mRNA transcript comprises quantifying changes in percent splice-in (dPSI) values for at least one transcript. In some embodiments, predicting translation of at least one selectively spliced mRNA transcript comprises RiboSeq and / or ribosome profiling.

In some embodiments of the neoantigen identification methods described herein, the method further comprises evaluating at least one peptide for the predicted major histocompatibility complex (MHC) binding. In some embodiments, the predicted MHC binding is determined by measuring the uncorrected affinity predicted binding strength of at least one peptide. In some embodiments, uncorrected affinity predicted binding intensities of about 500 nM or higher indicate MHC binding. In some embodiments, predicted MHC binding is determined by identifying a distribution of predicted binding intensities for a series of random peptides; and comparing the predicted binding strength of at least one peptide to that distribution. In some embodiments, the top 2% of the distribution shows MHC bonds with weak predicted bond strength. In some embodiments, the top 0.5% of the distribution shows MHC bonds with strong predicted bond strength.

In some embodiments of the neoantigen identification methods described herein, neoplastic cells are present in in vitro cell cultures. In some embodiments, neoplastic cells are obtained from the subject. In some embodiments, neoplastic cells are present in the subject.

Also included herein are, in various embodiments, (a) at least one neoantigen (eg, at least one neoantigen peptide or code thereof) using any of the exemplary identification methods disclosed herein. (MRNA); and (b) a pharmaceutically acceptable carrier, diluent, or adjuvant (eg, pharmaceutically acceptable carrier, diluent described herein) for at least one neoantigen. , Or an adjuvant) is also provided to make a neoantigen vaccine by formulation.

In some embodiments, the at least one neoantigen and / or antigenic moiety ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the at least one neoantigen and / or antigenic moiety ranges from about 15 to about 25 amino acids in length. In some embodiments, the at least one neoantigen and / or antigenic moiety ranges from about 10 to about 20 amino acids in length. In some embodiments, the at least one neoantigen and / or antigenic moiety does not exclusively overlap with a canonical peptide sequence (eg, any of the exemplary canonical peptide sequences underlined in Table 8). , Or not.

In some embodiments, the at least one neoantigen used in the vaccine is linked to a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. Toxoid.

Harboxydiene splicing regulator / ADC in combination with modified T cells (CAR-T):
In various embodiments, patients with cancer as described herein are harboxydiene splicing regulators, ADCs, or compositions and one or more modified tumor-targeted T cells (ie, CAR-T). ) Can be treated in combination. Accordingly, in various embodiments, the present disclosure comprises a subject having or suspected of having a neoplastic disorder in an effective amount of a harboxydiene splicing regulator, ADC, or harboxydiene splicing regulator or ADC. And provide a method of treating by administering a modified tumor targeting T cell (ie, CAR-T) to a subject. In various embodiments, the chimeric T cell receptor can be modified using an antigen recognition sequence that is reactive with the identified neoantigen.

For example, in various embodiments, the chimeric antigen-reactive T cell receptor (CAR) is modified to target changes in the extracellular domain of cell surface proteins induced by harboxidien splicing regulators or ADCs. Often, this is by initially identifying antibodies that recognize the cell surface-expressed neoantigen protein domain. The antigen recognition sequence of such antibody can then be fused to the T cell receptor domain for selective targeting and activation.

In various other embodiments, strategies are used to integrate the antigen presenting mechanism of tumor cells with neoantigens derived from harboxydiene splicing regulators or ADCs. In some embodiments, cells containing known high frequency HLA alleles (eg, HLA-A * 02: 01) can be treated with a harboxydiene splicing regulator, ADC, or composition. , Ligand mix identifies MHC1-bound neoantigens. In some embodiments, these peptides can be used to prime and / or expand T cells from healthy donors expressing the same HLA allele. In some embodiments, such T cells can be isolated and the T cell receptor (TCR) α and β chains sequenced to identify the cognate antigen recognition / variable region. In some embodiments, the cognate CAR can then be modified.

In some embodiments, the CAR sequence is cloned and expanded into a patient-derived T cell population using currently available protocols. In some embodiments, after treatment with a harboxydiene splicing regulator, ADC, or composition, modified T cells are infused and returned to the patient's circulation. After treatment with a harboxydiene splicing regulator, ADC, or composition, in some embodiments, tumor cells may begin to present antigen. In some embodiments, the modified T cell population can associate with and kill antigen-presenting tumor cells.

Other suitable modifications and adaptations of the methods of the present disclosure described herein are obvious to those of skill in the art and are suitable without departing from the scope of this disclosure or embodiments disclosed herein. It will be easily understood that this can be done with the equivalent. This disclosure has been described in detail here, but it will be more clearly understood by reference to the following examples, which are included for illustration purposes only and are not intended to be limiting.

Example 1
Methods for synthesizing the payloads, linkers, and conjugateable linker-payload (linker-drug, LH) compounds whose structures are shown in Tables 9-11 are described. The connectable linker-payload was used in the preparation of antibody-drug conjugates (ADCs). Exemplary ADCs are described in Examples 3-5.

1.1 Reagents and Materials The starting materials used in the following synthetic methods are either commercially available or readily prepared from known materials by standard methods. The disclosed conjugateable linker-payload can be prepared using the reactions and techniques described herein. Unless otherwise indicated, in the description of the synthetic method described below, all proposed reaction conditions include the solvent, reaction atmosphere, reaction temperature, time required for the experiment, and selection of work-up procedures for the reaction. It should be understood that it can be selected to be a standard condition. Those skilled in the art of organic synthesis will appreciate that the functional groups present in various parts of the molecule must be compatible with the proposed reagents and reactions. Substituents that are incompatible with the reaction conditions will be apparent to those of skill in the art and therefore alternative methods are indicated herein.

Preparative liquid chromatography-mass spectrometry (LC / MS) was performed under acidic mobile phase conditions using a Waters AutoPurification System and an XTerra MS C18 column (5 μm, 19 mm × 100 mm). Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz using a Varian instrument (Agilent Technologies). Microwave heating was performed using Biotage Emrys Liberator or Initiator microwave. Column chromatography was performed using Teledyne Isco Combiflash Rf200d. Solvent removal was performed using either a Buechi rotary evaporator or a Genevac centrifugal evaporator.

Term / Abbreviation: As used herein, the term "inactivated" means that the air in a reactor (eg, reactor, flask, glass reactor) is essentially water-free nitrogen. Or it means that it is replaced with an inert gas such as argon. The following abbreviations are used herein: DCM = dichloromethane, DMF = dimethylformamide, HPLC = high performance liquid chromatography, KHMDS = potassium bis (trimethylsilyl) amide, LC / MS = liquid chromatography-mass spectrometry, MeOH = Methanol, RT = room temperature, TBSCl = tert-butyldimethylsilyl chloride, THF = tetrahydrofuran, TLC = thin layer chromatography. Multiplicity is indicated using the following abbreviations: s = single line, d = double line, t = triple line, q = quadruple line, quint = quintuple line, sxt = six-fold line, m = multiplex. Line, dd = double line of double line, ddd = double line of double line, dt = double line of triple line, br s = wide single line.

LC / MS: Mobile phase = A (0.1% formic acid in H2O) and B (0.1% formic acid in acetonitrile). Radiant = B 5% -95%, 1.8 minutes. Column = Waters Accuracy BEH C18 column (1.7 μm, 2.1 × 50 mm).

ステップ１：発酵及びバイオコンバージョン
１０ｍＬのＳＹ－３２培地（１％Ｄ－グルコース、１％可溶性デンプン、０．５％バクトソイトン、０．５％酵母エキス、０．２％硫酸アンモニウム、０．２％ＮａＣｌ及び２．３％ＴＥＳ、ｐＨ８．０）が入った試験管内の第１の種培養物に、日本の土壌から分離されたサッカロトリクス属種（Ｓａｃｃｈａｒｏｔｈｒｉｘ ｓｐ．）ＥＡＳ－ＡＢ４５６４の２０パーセントグリセロールストック溶液を接種した。この第１の種培養物を２００ｒｐｍのレシプロシェーカーにおいて２８℃で２日間振盪した。発酵後、第１の種培養物を、各々１００ｍＬの新しいＳＹ－３２培地が入った三角フラスコ内の第２の滅菌種培養物に１％ｖ／ｖで接種した。第２の種培養物を２００ｒｐｍのロータリーシェーカー（Ｉｗａｓｈｉｙａ ｂｉｏｓｃｉｅｎｃｅ ＳＣ－１４４－ＧＲ）において２８℃で２日間振盪した。発酵後、２５０ｍＬの第２の培養物を、１０Ｌの新しいＳＹ－３２培地及び２ｍＬの消泡剤ＰＥ－Ｍが入った１５Ｌ発酵槽（Ｓａｎｋｉ ｓｅｉｋｉ ＭＡＴ－１５）に接種した。発酵は２８℃で撹拌及び曝気条件下（４５０ｒｐｍ、１０Ｌ／分）に行った。４８時間後、培養ブロスを３０００ｒｐｍで１０分間遠心した。上清の除去後、微生物ペレットに１０Ｌの２０ｍＭリン酸緩衝液（ｐＨ７．０）を加えて微生物細胞を洗浄した。この懸濁液を３０００ｒｐｍで１０分間遠心した。洗浄緩衝液の除去後、微生物ペレットに１０Ｌの反応緩衝液（１％Ｄ－グルコース、０．２％塩化マグネシウム六水和物、２．３％ＴＥＳ及び１．３ｇのハーボキシジエン、ｐＨ８．０）を加え、懸濁液を１５Ｌ発酵槽に移した。バイオコンバージョンは、撹拌及び曝気（４５０ｒｐｍ、１０Ｌ／分）しながら２８℃で６時間行った。 1.2 Preparation of ADL1-H1, ADL1-H2, ADL1-H3, and ADL1-H4 1.2.1 Overview-General Procedure 1

Step 1: Fermentation and bioconversion 10 mL of SY-32 medium (1% D-glucose, 1% soluble starch, 0.5% bactosoyton, 0.5% yeast extract, 0.2% ammonium sulfate, 0.2% NaCl and A 20% glycerol stock solution of Saccarotrix sp. EAS-AB4564 isolated from Japanese soil in a first seed culture in vitro containing 2.3% TES, pH 8.0). Was inoculated. The first seed culture was shaken at 28 ° C. for 2 days in a 200 rpm reciprocating shaker. After fermentation, the first seed culture was inoculated at 1% v / v into the second sterile seed culture in an Erlenmeyer flask, each containing 100 mL of fresh SY-32 medium. The second seed culture was shaken at 28 ° C. for 2 days in a 200 rpm rotary shaker (Iwashiya bioscience SC-144-GR). After fermentation, 250 mL of the second culture was inoculated into a 15 L fermenter (Sanki seiki MAT-15) containing 10 L of fresh SY-32 medium and 2 mL of antifoaming agent PE-M. Fermentation was carried out at 28 ° C. under stirring and aeration conditions (450 rpm, 10 L / min). After 48 hours, the culture broth was centrifuged at 3000 rpm for 10 minutes. After removal of the supernatant, 10 L of 20 mM phosphate buffer (pH 7.0) was added to the microbial pellet to wash the microbial cells. The suspension was centrifuged at 3000 rpm for 10 minutes. After removal of wash buffer, add 10 L of reaction buffer (1% D-glucose, 0.2% magnesium chloride hexahydrate, 2.3% TES and 1.3 g harboxydiene, pH 8.0) to the microbial pellet. In addition, the suspension was transferred to a 15 L fermenter. Bioconversion was performed at 28 ° C. for 6 hours with stirring and aeration (450 rpm, 10 L / min).

Isolation of 5-Hydroxyharboxidiene XAD-7HP (400 g) was added to the above mixture (10 L) and the mixture was stirred at 300 rpm for 30 minutes (EYELA MAZELA Z). After stirring, separation of XAD-7HP was determined using a staining and inspection sieve (0.25 mm aperture and 0.16 mm wire diameter). The same operation was repeated. The collected XAD-7HP was extracted with 1 L of acetone and the solvent was removed in vacuo. When the extract is purified by a gradient elution method (YMC-DispoPack AT ODS-25 120 g, 25-55% acetonitrile in water with 0.1% formic acid, 15 minutes) by reverse phase MPLC (Yamazen EPCLC-W-prep 2XY). , 5-OH harboxidiene (397.47 mg) was obtained with a conversion yield of 29.5%. ^{1 1} H-NMR and mass spectrometry data were in agreement with the literature values. For example, European Patent No. 0781772 B1 and Ghosh et al. (2014) Org. Let. See 16: 3154-57.

０℃でＴＨＦ（２ｍＬ）及びＭｅＯＨ（０．５ｍＬ）中の２－（（２Ｒ，４Ｒ，５Ｓ，６Ｓ）－４－ヒドロキシ－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）酢酸（４５ｍｇ、０．０９９ｍｍｏｌ）の溶液に、トリメチルシリルジアゾメタン（ヘキサン中２．０Ｍ、０．１４８ｍＬ、０．２９７ｍｍｏｌ）を滴下して加えた。次に得られた混合物を室温になるまで徐々に加温し、１時間撹拌した後、０℃に冷却した。次に酢酸（０．０１７ｍＬ、０．２９７ｍｍｏｌ）を加え、３０分間撹拌した。次にこの混合物をＡｃＯＥｔ及び飽和重炭酸ナトリウム水溶液で希釈した。有機層を分離し、水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。分離された残渣をシリカゲルクロマトグラフィーにより精製して、メチル２－（（２Ｒ，４Ｒ，５Ｓ，６Ｓ）－４－ヒドロキシ－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）アセテートを無色の油として得た（３６．１ｍｇ、７８％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８６（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）１．２０－１．２４（ｍ，１Ｈ）１．２６（ｓ，３Ｈ）１．３１－１．３４（ｍ，３Ｈ）１．４０－１．５９（ｍ，２Ｈ）１．６３（ｂｒ．ｓ．，２Ｈ）１．６９（ｓ，３Ｈ）１．８７（ｄｄ，Ｊ＝１３．６６，４．８８Ｈｚ，１Ｈ）２．０３（ｄｄｄ，Ｊ＝１０．３７，４．０２，２．２０Ｈｚ，１Ｈ）２．４２（ｄｄ，Ｊ＝１５．６１，６．３４Ｈｚ，２Ｈ）２．５４（ｄ，Ｊ＝９．８Ｈｚ，２Ｈ）２．６２（ｄｄ，Ｊ＝１５．６，６．３Ｈｚ，１Ｈ）２．９５（ｔ，Ｊ＝５．３７Ｈｚ，１Ｈ）３．３３－３．４４（ｍ，２Ｈ）３．５２（ｓ，３Ｈ）３．６５（ｓ，３Ｈ）３．７９－３．９０（ｍ，２Ｈ）５．４５（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８８（ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．２１（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． Step 2: Methyl 2-((2R, 4R, 5S, 6S) -4-hydroxy-6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) ) -4-Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran- 2-Il) Synthesis of acetate

THF at 0 ° C. (2 mL) and MeOH (0.5 mL) in 2 - ((2R, 4R, 5S, 6S) -4- hydroxy -6 - ((S, 2E, 4E) -7 - ((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2- To a solution of -5-methyltetrahydro-2H-pyran-2-yl) acetic acid (45 mg, 0.099 mmol), trimethylsilyldiazomethane (2.0 M in hexane, 0.148 mL, 0.297 mmol) is added dropwise. rice field. The resulting mixture was then gradually warmed to room temperature, stirred for 1 hour and then cooled to 0 ° C. Next, acetic acid (0.017 mL, 0.297 mmol) was added, and the mixture was stirred for 30 minutes. The mixture was then diluted with AcOEt and saturated aqueous sodium bicarbonate solution. The organic layer was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The separated residue was purified by silica gel chromatography to perform methyl 2-((2R, 4R, 5S, 6S) -4-hydroxy-6-((S, 2E, 4E) -7-((2R, 3R)). -3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-Methyltetrahydro-2H-pyran-2-yl) acetate was obtained as a colorless oil (36.1 mg, 78% yield).
^{1 1} H NMR (400MHz, chloroform-d) 0.80 (d, J = 6.83Hz, 3H) 0.86 (d, J = 6.83Hz, 3H) 1.03 (d, J = 6.83Hz, 3H) 1.16 (d, J = 6.3Hz, 3H) 1.20-1.24 (m, 1H) 1.26 (s, 3H) 1.31-1.34 (m, 3H) 1. 40-1.59 (m, 2H) 1.63 (br. S., 2H) 1.69 (s, 3H) 1.87 (dd, J = 13.66, 4.88Hz, 1H) 2.03 (Ddd, J = 10.37, 4.02, 2.20Hz, 1H) 2.42 (dd, J = 15.61, 6.34Hz, 2H) 2.54 (d, J = 9.8Hz, 2H) ) 2.62 (dd, J = 15.6, 6.3Hz, 1H) 2.95 (t, J = 5.37Hz, 1H) 3.33-3.44 (m, 2H) 3.52 (s) , 3H) 3.65 (s, 3H) 3.79-3.90 (m, 2H) 5.45 (dd, J = 15.12, 8.78Hz, 1H) 5.88 (d, J = 11) .22Hz, 1H) 6.21 (dd, J = 15.12, 10.73Hz, 1H).

Step 3: Carbamate synthesis Methyl 2-((2R, 4R, 5S, 6S) -4-hydroxy-6-((S, 2E, 4E) -7-((2R, 4E) -7-((2R, 4R, 4E) -7-((2R, 4R, 4E) -7-((2R, 4R, 4E) -7-((S, 2E, 4E) -7-((2R, 4R, 5S, 6S) -4-hydroxy-6-((S, 2E, 4E) -7-((2R, 4R, 4E) -7-((2R, 4R, 4E) -7-" , 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2 -Il) -5-Methyltetrahydro-2H-pyran-2-yl) acetate (1.0 eq, 0.075 mmol), 4-nitrophenyl carbonochloride (2 eq), Hunig base (0.065 mL, 4) DMAP (0.05 eq) was added to the mixture (0.5 eq). The mixture was then warmed to room temperature and stirred for 16 hours. Piperazine (10 eq) was added to this mixture and the resulting mixture was stirred for an additional hour. The mixture was then diluted with DCM, the organic layer was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography and then by amino-functionalized silica gel chromatography to give the desired product.

Step 4: Carboxylic acid synthesis Add aqueous sodium hydroxide solution (1.0 eq, 2N) to the mixture of methyl esters (1.0 eq, 0.039 mmol) obtained from step 3 in MeOH (0.02 Mml). rice field. The mixture was heated to 40 ° C. and stirred at that temperature for 4 hours. The resulting mixture was then cooled to 0 ° C. and neutralized with aqueous hydrochloric acid (200 μL, 2N). The mixture is then concentrated in vacuo and the resulting residue is purified by preparative HPLC (H2O / MeCN / HCOOH = 80/20 / 0.1-60 / 40 / 0.1) to give the desired product. Obtained.

セクション１．２．１のステップ３のとおり表題化合物を合成して淡黄色の油を得た（２２．３ｍｇ、５２％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．７１（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．２０－１．２５（ｍ，１Ｈ）１．２６（ｓ，３Ｈ）１．３７（ｑ，Ｊ＝１１．５５Ｈｚ，１Ｈ）１．５１（ｄｔ，Ｊ＝８．９０，６．５２Ｈｚ，１Ｈ）１．６３－１．６７（ｍ，１Ｈ）１．６９（ｓ，３Ｈ）１．８３－２．０７（ｍ，１０Ｈ）２．１１－２．１７（ｍ，１Ｈ）２．３６－２．４５（ｍ，２Ｈ）２．５１－２．６２（ｍ，２Ｈ）２．８１（ｓ，４Ｈ）２．９５（ｔ，Ｊ＝５．１２Ｈｚ，１Ｈ）３．４０－３．４９（ｍ，４Ｈ）３．５２（ｓ，３Ｈ）３．６５（ｓ，３Ｈ）３．８０－３．９４（ｍ，２Ｈ）４．５６（ｔｄ，Ｊ＝１０．９８，４．３９Ｈｚ，１Ｈ）５．４６（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）６．２１（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． 1.2.2 Synthesis of H1 (2S, 3S, 4R, 6R) -2-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4) -Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -6- (2-methoxy-2-oxoethyl)- 3-Methyltetrahydro-2H-pyran-4-ylpiperazin-1-carboxylate

The title compound was synthesized as in step 3 of Section 1.2.1 to give a pale yellow oil (22.3 mg, 52% yield).
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.71 (d, J = 6.83 Hz, 3H) 0.85 (d, J = 6.8 Hz, 3H) 1.03 (d, J = 6. 83Hz, 3H) 1.16 (d, J = 6.8Hz, 3H) 1.20-1.25 (m, 1H) 1.26 (s, 3H) 1.37 (q, J = 11.55Hz, 1H) 1.51 (dt, J = 8.90, 6.52Hz, 1H) 1.63-1.67 (m, 1H) 1.69 (s, 3H) 1.83-2.07 (m, 10H) 2.11-2.17 (m, 1H) 2.36-2.45 (m, 2H) 2.51-2.62 (m, 2H) 2.81 (s, 4H) 2.95 ( t, J = 5.12Hz, 1H) 3.40-3.49 (m, 4H) 3.52 (s, 3H) 3.65 (s, 3H) 3.80-3.94 (m, 2H) 4.56 (td, J = 10.98, 4.39Hz, 1H) 5.46 (dd, J = 15.12, 8.78Hz, 1H) 5.90 (d, J = 10.24Hz, 1H) 6.21 (dd, J = 15.12, 10.73Hz, 1H).

セクション１．２．１のステップ４のとおり表題化合物を合成して無色の油を得た（１６．０ｍｇ、７３％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），５６７．７７［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．７２（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）１．１７（ｄｄ，Ｊ＝１３．２，１１．２Ｈｚ，１Ｈ）１．２５（ｓ，３Ｈ）１．３６（ｑ，Ｊ＝１１．４Ｈｚ，１Ｈ）１．４５－１．５０（ｍ，２Ｈ）１．６８（ｓ，３Ｈ）１．９０（ｄｄ，Ｊ＝１３．４２，４．１５Ｈｚ，１Ｈ）２．１４（ｄｄ，Ｊ＝１１．７１，３．９０Ｈｚ，１Ｈ）２．３６－２．５２（ｍ，３Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．９５（ｄｄ，Ｊ＝５．８５，４．３９Ｈｚ，１Ｈ）３．１７（ｂｒ．ｓ，４Ｈ）３．４６－３．５３（ｍ，４Ｈ）３．５６－３．６０（ｍ，１Ｈ）３．６９ ｎｒ．ｓ，４Ｈ）３．７４－３．８２（ｍ，２Ｈ）３．８５－３．９２（ｍ，１Ｈ）４．５８（ｔｄ，Ｊ＝１０．４９，４．３９Ｈｚ，１Ｈ）５．４９（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．９４（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）． 2-((2R, 4R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-) Methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyl-4-((piperazine-1-carbonyl) oxy) tetrahydro -2H-pyran-2-yl) acetic acid (H1)

The title compound was synthesized as in step 4 of Section 1.2.1 to give a colorless oil (16.0 mg, 73% yield). LC / MS (ESI, m / z), 567.77 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.72 (d, J = 6.3 Hz, 3H) 0.80 (d, J = 6.83 Hz, 3H) 1.03 (d, J = 6) .3Hz, 3H) 1.08 (d, J = 6.3Hz, 3H) 1.17 (dd, J = 13.2, 11.2Hz, 1H) 1.25 (s, 3H) 1.36 (q) , J = 11.4Hz, 1H) 1.45-1.50 (m, 2H) 1.68 (s, 3H) 1.90 (dd, J = 13.42, 4.15Hz, 1H) 2.14 (Dd, J = 11.71, 3.90Hz, 1H) 2.36-2.52 (m, 3H) 2.63 (d, J = 9.27Hz, 1H) 2.95 (dd, J = 5) .85, 4.39Hz, 1H) 3.17 (br.s, 4H) 3.46-3.53 (m, 4H) 3.56-3.60 (m, 1H) 3.69 nr. s, 4H) 3.74-3.82 (m, 2H) 3.85-3.92 (m, 1H) 4.58 (td, J = 10.49, 4.39Hz, 1H) 5.49 ( dd, J = 15.12, 8.78Hz, 1H) 5.94 (d, J = 10.73Hz, 1H) 6.28 (dd, J = 14.88, 10.98Hz, 1H).

セクション１．２．１のステップ３のとおり表題化合物を合成した（３０．３ｍｇ、６０％収率）。
^１Ｈ ＮＭＲ（５００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．７１（ｄ，Ｊ＝６．１１Ｈｚ，３Ｈ）０．８４（ｄ，Ｊ＝６．７３Ｈｚ，３Ｈ）１．００－１．０５（ｍ，４Ｈ）１．１６（ｄ，Ｊ＝６．１１Ｈｚ，３Ｈ）１．１９－１．２４（ｍ，１Ｈ）１．２６（ｓ，３Ｈ）１．３２－１．４０（ｍ，１Ｈ）１．５１（ｄｔ，Ｊ＝９．１７，６．４２Ｈｚ，１Ｈ）１．６３－１．６７（ｍ，１Ｈ）１．６９（ｓ，３Ｈ）１．７３－１．８１（ｍ，３Ｈ）１．８３－１．９０（ｍ，２Ｈ）２．０７－２．１８（ｍ，１Ｈ）２．４１（ｄｄ，Ｊ＝１５．２８，６．１１Ｈｚ，２Ｈ）２．５１－２．６０（ｍ，２Ｈ）２．８２－２．９５（ｍ，５Ｈ）３．４１－３．４９（ｍ，４Ｈ）３．５１（ｓ，３Ｈ）３．６４（ｓ，３Ｈ）３．７９－３．９５（ｍ，２Ｈ）４．５７（ｔｄ，Ｊ＝１０．７０，４．２８Ｈｚ，１Ｈ）５．４５（ｄｄ，Ｊ＝１４．９８，８．８６Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１１．００Ｈｚ，１Ｈ）６．２０（ｄｄ，Ｊ＝１４．９８，１０．７０Ｈｚ，１Ｈ）． 1.2.3 Synthesis of H2 (2S, 3S, 4R, 6R) -2-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4) -Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -6- (2-methoxy-2-oxoethyl)- 3-Methyltetrahydro-2H-pyran-4-yl 1,4-diazepan-1-carboxylate

The title compound was synthesized as in step 3 of Section 1.2.1 (30.3 mg, 60% yield).
^{1 1} H NMR (500 MHz, chloroform-d) δ ppm 0.71 (d, J = 6.11 Hz, 3H) 0.84 (d, J = 6.73 Hz, 3H) 1.00-1.05 (m, 4H) 1.16 (d, J = 6.11Hz, 3H) 1.19-1.24 (m, 1H) 1.26 (s, 3H) 1.32-1.40 (m, 1H) 1. 51 (dt, J = 9.17, 6.42 Hz, 1H) 1.63-1.67 (m, 1H) 1.69 (s, 3H) 1.73-1.81 (m, 3H) 1. 83-1.90 (m, 2H) 2.07-2.18 (m, 1H) 2.41 (dd, J = 15.28, 6.11Hz, 2H) 2.51-2.60 (m, 2H) 2.82-2.95 (m, 5H) 3.41-3.49 (m, 4H) 3.51 (s, 3H) 3.64 (s, 3H) 3.79-3.95 ( m, 2H) 4.57 (td, J = 10.70, 4.28Hz, 1H) 5.45 (dd, J = 14.98, 8.86Hz, 1H) 5.90 (d, J = 11. 00Hz, 1H) 6.20 (dd, J = 14.98, 10.70Hz, 1H).

セクション１．２．１のステップ４のとおり表題化合物を合成して無色の油を得た（２０．９ｍｇ、７１％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），５８１．８１［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．７４（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１２－１．２３（ｍ，１Ｈ）１．２５（ｓ，３Ｈ）１．３１－１．４１（ｍ，１Ｈ）１．４１－１．５３（ｍ，１Ｈ）１．６３－１．７４（ｍ，４Ｈ）１．８４－１．９３（ｍ，１Ｈ）２．０４（ｂｒ．ｓ．，２Ｈ）２．１１－２．２１（ｍ，１Ｈ）２．３４－２．５４（ｍ，３Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．９０－２．９８（ｍ，２Ｈ）３．２１－３．３６（ｍ，７Ｈ）３．５０（ｓ，３Ｈ）３．５３－３．６６（ｍ，２Ｈ）３．６９－３．７９（ｍ，３Ｈ）３．８３－３．９５（ｍ，１Ｈ）４．５７（ｂｒ．ｓ．，１Ｈ）５．４９（ｄｄ，Ｊ＝１４．８８，９．０３Ｈｚ，１Ｈ）５．９４（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２４－６．３６（ｍ，１Ｈ）． 2-((2R, 4R, 5S, 6S) -4-((1,4-diazepan-1-carbonyl) oxy) -6-((S, 2E, 4E) -7-((2R, 3R)-) 3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl)- 5-Methyltetrahydro-2H-pyran-2-yl) acetic acid (H2)

The title compound was synthesized as in step 4 of Section 1.2.1 to give a colorless oil (20.9 mg, 71% yield). LC / MS (ESI, m / z), 581.81 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.74 (d, J = 6.34 Hz, 3H) 0.80 (d, J = 7.32 Hz, 3H) 1.03 (d, J = 6) .34Hz, 3H) 1.08 (d, J = 6.34Hz, 3H) 1.12-1.23 (m, 1H) 1.25 (s, 3H) 1.31-1.41 (m, 1H) ) 1.41-1.53 (m, 1H) 1.63-1.74 (m, 4H) 1.84-1.93 (m, 1H) 2.04 (br.s., 2H) 2. 11-2-21 (m, 1H) 2.34-2.54 (m, 3H) 2.63 (d, J = 9.27Hz, 1H) 2.90-2.98 (m, 2H) 3. 21-3.36 (m, 7H) 3.50 (s, 3H) 3.53-3.66 (m, 2H) 3.69-3.79 (m, 3H) 3.83-3.95 ( m, 1H) 4.57 (br.s., 1H) 5.49 (dd, J = 14.88, 9.03Hz, 1H) 5.94 (d, J = 10.73Hz, 1H) 6.24 -6.36 (m, 1H).

セクション１．２．１のステップ３のとおり表題化合物を合成して無色の油を得た（９．６ｍｇ、１９％収率）。
１Ｈ ＮＭＲ（５００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．７１（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．７３Ｈｚ，３Ｈ）０．９９－１．０５（ｍ，４Ｈ）１．１７（ｄ，Ｊ＝６．１１Ｈｚ，４Ｈ）１．２７（ｓ，３Ｈ）１．３２－１．４５（ｍ，１Ｈ）１．４９－１．６８（ｍ，６Ｈ）１．７０（ｓ，３Ｈ）１．８８（ｄｄ，Ｊ＝１３．４５，４．８９Ｈｚ，１Ｈ）２．００－２．０８（ｍ，１Ｈ）２．１０－２．１７（ｍ，１Ｈ）２．３８－２．４７（ｍ，５Ｈ）２．５２－２．６１（ｍ，２Ｈ）２．９４（ｔ，Ｊ＝５．５０Ｈｚ，１Ｈ）３．１７－３．２６（ｍ，１Ｈ）３．４３（ｄ，Ｊ＝１０．４Ｈｚ，２Ｈ）３．５２（ｓ，４Ｈ）３．６５（ｓ，３Ｈ）３．７８－３．８６（ｍ，１Ｈ）３．８７－３．９５（ｍ，１Ｈ）４．５１－４．５７（ｍ，１Ｈ）５．４５（ｄｄ，Ｊ＝１４．９８，８．８６Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１０．３９Ｈｚ，１Ｈ）６．２１（ｄｄ，Ｊ＝１４．９８，１０．７０Ｈｚ，１Ｈ）． 1.2.4 Synthesis of H3 (2S, 3S, 4R, 6R) -2-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4) -Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -6- (2-methoxy-2-oxoethyl)- 3-Methyltetrahydro-2H-pyran-4-yl 3- (methylamino) pyrrolidine-1-carboxylate

The title compound was synthesized as in step 3 of Section 1.2.1 to give a colorless oil (9.6 mg, 19% yield).
1H NMR (500MHz, chloroform-d) δ ppm 0.71 (d, J = 6.3Hz, 3H) 0.85 (d, J = 6.73Hz, 3H) 0.99-1.05 (m, 4H) ) 1.17 (d, J = 6.11Hz, 4H) 1.27 (s, 3H) 1.32-1.45 (m, 1H) 1.49-1.68 (m, 6H) 1.70 (S, 3H) 1.88 (dd, J = 13.45, 4.89Hz, 1H) 2.00-2.08 (m, 1H) 2.10-2.17 (m, 1H) 2.38 -2.47 (m, 5H) 2.52-2.61 (m, 2H) 2.94 (t, J = 5.50Hz, 1H) 3.17-3.26 (m, 1H) 3.43 (D, J = 10.4Hz, 2H) 3.52 (s, 4H) 3.65 (s, 3H) 3.78-3.86 (m, 1H) 3.87-3.95 (m, 1H) ) 4.51-4.57 (m, 1H) 5.45 (dd, J = 14.98, 8.86Hz, 1H) 5.90 (d, J = 10.39Hz, 1H) 6.21 (dd) , J = 14.98, 10.70Hz, 1H).

セクション１．２．１のステップ４のとおり表題化合物を合成して無色の油を得た（９．８ｍｇ、定量的収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），５８１．８１［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．７１（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０２（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．２２－１．２４（ｍ，１Ｈ）１．２６（ｓ，３Ｈ）１．３１－１．４２（ｍ，１Ｈ）１．４５－１．６６（ｍ，４Ｈ）１．６９（ｓ，３Ｈ）１．８７（ｄｄ，Ｊ＝１３．６６，４．８８Ｈｚ，１Ｈ）２．０２－２．２０（ｍ，２Ｈ）２．３８－２．５１（ｍ，４Ｈ）２．５１－２．６０（ｍ，２Ｈ）２．９４（ｔ，Ｊ＝５．１２Ｈｚ，１Ｈ）３．０８－３．２６（ｍ，２Ｈ）３．３３－３．４０（ｍ，１Ｈ）３．４３（ｄ，Ｊ＝９．７６Ｈｚ，２Ｈ）３．５１（ｓ，３Ｈ）３．６４（ｓ，３Ｈ）３．８３－３．９４（ｍ，１Ｈ）４．５１－４．５９（ｍ，１Ｈ）５．４５（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８９（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２０（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． 2-((2R, 4R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-) Methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyl-4-((3- (methylamino) pyrrolidine-1) -Carbonyl) oxy) tetrahydro-2H-pyran-2-yl) acetic acid (H3)

The title compound was synthesized as in step 4 of Section 1.2.1 to give a colorless oil (9.8 mg, quantitative yield). LC / MS (ESI, m / z), 581.81 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.71 (d, J = 6.34 Hz, 3H) 0.85 (d, J = 6.83 Hz, 3H) 1.02 (d, J = 6) .34Hz, 3H) 1.16 (d, J = 6.34Hz, 3H) 1.22-1.24 (m, 1H) 1.26 (s, 3H) 1.31-1.42 (m, 1H) ) 1.45-1.66 (m, 4H) 1.69 (s, 3H) 1.87 (dd, J = 13.66, 4.88Hz, 1H) 2.02-2.20 (m, 2H) ) 2.38-2.51 (m, 4H) 2.51-2.60 (m, 2H) 2.94 (t, J = 5.12Hz, 1H) 3.08-3.26 (m, 2H) ) 3.33-3.40 (m, 1H) 3.43 (d, J = 9.76Hz, 2H) 3.51 (s, 3H) 3.64 (s, 3H) 3.83-3.94 (M, 1H) 4.51-4.59 (m, 1H) 5.45 (dd, J = 15.12, 8.78Hz, 1H) 5.89 (d, J = 10.73Hz, 1H) 6 .20 (dd, J = 15.12, 10.73Hz, 1H).

セクション１．２．１のステップ３（１３．８ｍｇ、９１％収率）及びステップ４のとおり表題化合物を合成して、白色のアモルファス固体（ａｒｍｏｐｈｏｕｓ ｓｏｌｉｄ）を得た（４．５８ｍｇ、７６％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），５８１．５５［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．７１（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．２６（ｓ，４Ｈ）１．４８－１．５７（ｍ，１Ｈ）１．６１－１．６８（ｍ，１Ｈ）１．８９（ｄｄ，Ｊ＝１３．６６，４．３９Ｈｚ，１Ｈ）２．２０（ｂｒ ｄ，Ｊ＝８．２９Ｈｚ，１Ｈ）２．３０（ｓ，４Ｈ）２．３３－２．５１（ｍ，６Ｈ）２．５２－２．５７（ｍ，２Ｈ）２．５７－２．６３（ｍ，１Ｈ）２．７８－３．２１（ｍ，９Ｈ）３．３５－３．６６（ｍ，９Ｈ）３．７０－３．９９（ｍ，２Ｈ）４．５２（ｂｒ ｄ，Ｊ＝３．９０Ｈｚ，１Ｈ）５．４１－５．５８（ｍ，１Ｈ）５．９２（ｂｒ ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．１３－６．２９（ｍ，１Ｈ）． 1.2.5 Synthesis of H12

The title compound was synthesized as described in Step 3 (13.8 mg, 91% yield) and Step 4 of Section 1.2.1 to give a white amorphous solid (4.58 mg, 76% yield). rate). LC / MS (ESI, m / z), 581.55 [M + H] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.71 (d, J = 6.83Hz, 3H) 0.85 (d, J = 6.83Hz, 3H) 1.03 (d, J = 6.34Hz) , 3H) 1.16 (d, J = 6.34Hz, 3H) 1.26 (s, 4H) 1.48-1.57 (m, 1H) 1.61-1.68 (m, 1H) 1 .89 (dd, J = 13.66, 4.39Hz, 1H) 2.20 (br d, J = 8.29Hz, 1H) 2.30 (s, 4H) 2.33-2.51 (m, 6H) 2.52-2.57 (m, 2H) 2.57-2.63 (m, 1H) 2.78-3.21 (m, 9H) 3.35-3.66 (m, 9H) 3.70-3.99 (m, 2H) 4.52 (br d, J = 3.90Hz, 1H) 5.41-5.58 (m, 1H) 5.92 (br d, J = 11. 22Hz, 1H) 6.13-6.29 (m, 1H).

ステップ１：２，５－ジオキソピロリジン－１－イル２－（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）アセテート

０℃でＤＣＭ（１０ｍｌ）中の２－（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）酢酸（ハーボキシジエン、３００ｍｇ、０．６８４ｍｍｏｌ）の混合物にＤＣＣ（３１０ｍｇ、１．５０５ｍｍｏｌ）を加え、得られた混合物を１５分間撹拌した。次に、この混合物にＮ－ヒドロキシコハク酸アミド（１７３ｍｇ、１．５０５ｍｍｏｌ）を加えた。得られた混合物（ｍｉｘｔｕｘｅ）を室温に加温し、１６時間撹拌した。続いて、混合物をセライトでろ過し、ろ過ケーキをＤＣＭで洗浄した。ろ液を真空濃縮し、得られた残渣をシリカゲルクロマトグラフィー（１２ｇ、ヘプタン／ＡｃＯＥｔ＝５０／５０～０／１００）により精製して、表題化合物を無色（ｃｏｌｏｒｏｌｅｓｓ）の固体として得た（３５７ｍｇ、９７％収率）。
^１Ｈ ＮＭＲ（５００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．７３Ｈｚ，３Ｈ）０．８３－０．８９（ｍ，３Ｈ）１．０３（ｄ，Ｊ＝６．７３Ｈｚ，３Ｈ）１．１７（ｄ，Ｊ＝６．１Ｈｚ，３Ｈ）１．１９－１．２５（ｍ，４Ｈ）１．２７（ｓ，３Ｈ）１．３９－１．５６（ｍ，３Ｈ）１．５９（ｓ，３Ｈ）１．７０（ｓ，３Ｈ）１．７９－１．９３（ｍ，３Ｈ）２．３６－２．４３（ｍ，１Ｈ）２．５２－２．５５（ｍ，２Ｈ）２．７０（ｄｄ，Ｊ＝１５．２８，７．３４Ｈｚ，１Ｈ）２．８１（ｂｒ．ｓ．，３Ｈ）２．８３－２．９０（ｍ，１Ｈ）２．９５（ｔ，Ｊ＝５．５０Ｈｚ，１Ｈ）３．３４（ｄ，Ｊ＝９．７８Ｈｚ，１Ｈ）３．５２（ｓ，３Ｈ）３．７９－３．８７（ｍ，２Ｈ）５．４３（ｄｄ，Ｊ＝１５．２８，９．１７Ｈｚ，１Ｈ）５．８８（ｄ，Ｊ＝１１．００Ｈｚ，１Ｈ）６．２１－６．２６（ｍ，１Ｈ）． 1.2.6 Synthesis of H4 H4 was synthesized according to Scheme 2:

Step 1: 2,5-Dioxopyrrolidine-1-yl 2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R) -3-((2R, 3R) -7- , 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro- 2H-pyran-2-yl) acetate

2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R)) in DCM (10 ml) at 0 ° C. -4-Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2 DCC (310 mg, 1.505 mmol) was added to a mixture of -yl) acetic acid (harboxydiene, 300 mg, 0.684 mmol) and the resulting mixture was stirred for 15 minutes. Next, N-hydroxysuccinamide (173 mg, 1.505 mmol) was added to this mixture. The obtained mixture (mixtune) was heated to room temperature and stirred for 16 hours. Subsequently, the mixture was filtered through Celite and the filtered cake was washed with DCM. The filtrate was concentrated in vacuo and the resulting residue was purified by silica gel chromatography (12 g, heptane / AcOEt = 50/50 to 0/100) to give the title compound as a colorless solid (357 mg, 97% yield).
^{1 1} H NMR (500 MHz, chloroform-d) δ ppm 0.66 (d, J = 6.73 Hz, 3H) 0.83-0.89 (m, 3H) 1.03 (d, J = 6.73 Hz, 3H) 1.17 (d, J = 6.1Hz, 3H) 1.19-1.25 (m, 4H) 1.27 (s, 3H) 1.39-1.56 (m, 3H) 1. 59 (s, 3H) 1.70 (s, 3H) 1.79-1.93 (m, 3H) 2.36-2.43 (m, 1H) 2.52-2.55 (m, 2H) 2.70 (dd, J = 15.28, 7.34Hz, 1H) 2.81 (br.s., 3H) 2.83-2.90 (m, 1H) 2.95 (t, J = 5) .50Hz, 1H) 3.34 (d, J = 9.78Hz, 1H) 3.52 (s, 3H) 3.79-3.87 (m, 2H) 5.43 (dd, J = 15.28) , 9.17Hz, 1H) 5.88 (d, J = 11.00Hz, 1H) 6.21-6.26 (m, 1H).

ＤＭＦ（２ｍＬ）中のステップ１で得られた化合物（４０ｍｇ、０．０７５ｍｍｏｌ）と（Ｓ）－２－アミノ－５－ヒドロキシペンタン酸（１９．８８ｍｇ、０．１４９ｍｍｏｌ）との混合物にＤＩＰＥＡ（０．０６５ｍＬ、０．３７３ｍｍｏｌ）を加えた。得られた混合物を室温で４時間撹拌し、次にＡｃＯＥｔで希釈した。有機層を分離し、水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。得られた残渣をステップ３で更なる精製なしに使用した。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．１５（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）１．１９－１．２３（ｍ，２Ｈ）１．２７（ｓ，３Ｈ）１．４０－１．６１（ｍ，６Ｈ）１．７２（ｓ，３Ｈ）１．８２－１．９３（ｍ，３Ｈ）２．３７－２．４３（ｍ，２Ｈ）２．５６（ｄ，Ｊ＝９．８Ｈｚ，１Ｈ）２．９９（ｔ，Ｊ＝５．３Ｈｚ，１Ｈ）３．３３（ｄ，Ｊ＝９．８Ｈｚ，１Ｈ）３．５２（ｓ，３Ｈ）３．５４－３．７０（ｍ，４Ｈ）３．８１－３．８７（ｍ，２Ｈ）４．５６（ｄ，Ｊ＝５．４Ｈｚ，１Ｈ）５．４８（ｄｄ，Ｊ＝１５．１２，８．２９Ｈｚ，１Ｈ）５．８９（ｄ，Ｊ＝１１．２Ｈｚ，１Ｈ）６．２２（ｄｄ，Ｊ＝１５．１，１０．７Ｈｚ，１Ｈ）７．３０（ｄ，Ｊ＝７．３Ｈｚ １Ｈ）． Step 2: (S) -5-Hydroxy-2- (2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R) , 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro- 2H-pyran-2-yl) acetamide) pentanic acid

DIPEA (0) in a mixture of the compound (40 mg, 0.075 mmol) obtained in step 1 in DMF (2 mL) and (S) -2-amino-5-hydroxypentanoic acid (19.88 mg, 0.149 mmol). .065 mL, 0.373 mmol) was added. The resulting mixture was stirred at room temperature for 4 hours and then diluted with AcOEt. The organic layer was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was used in step 3 without further purification.
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.66 (d, J = 6.3 Hz, 3H) 0.85 (d, J = 6.8 Hz, 3H) 1.03 (d, J = 6. 8Hz, 3H) 1.15 (d, J = 6.3Hz, 3H) 1.19-1.23 (m, 2H) 1.27 (s, 3H) 1.40-1.61 (m, 6H) 1.72 (s, 3H) 1.82-1.93 (m, 3H) 2.37-2.43 (m, 2H) 2.56 (d, J = 9.8Hz, 1H) 2.99 ( t, J = 5.3Hz, 1H) 3.33 (d, J = 9.8Hz, 1H) 3.52 (s, 3H) 3.54-3.70 (m, 4H) 3.81-3. 87 (m, 2H) 4.56 (d, J = 5.4Hz, 1H) 5.48 (dd, J = 15.12, 8.29Hz, 1H) 5.89 (d, J = 11.2Hz, 1H) 6.22 (dd, J = 15.1, 10.7Hz, 1H) 7.30 (d, J = 7.3Hz 1H).

０℃でＴＨＦ（２ｍＬ）及びメタノール（０．５ｍＬ）中のステップ２から得られた化合物（３５ｍｇ、０．０６３ｍｍｏｌ）の混合物にトリメチルシリルジアゾメタン（ヘキサン中２．０Ｍ、０．０９５ｍＬ、０．１９ｍｍｏｌ）を加えた。次にこの混合物を室温に加温し、１時間撹拌した。続いて、この混合物を０℃に冷却し、酢酸を加えることによりクエンチした。得られた混合物を３０分間撹拌し、次に真空濃縮した。得られた残渣をシリカゲルクロマトグラフィー（１２ｇ、ヘプタン／ＡｃＯＥｔ＝９０／１０～３０／７０）により精製して、表題化合物を得た（１５．７ｍｇ、４４％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．１９－１．２３（ｍ，３Ｈ）１．２７（ｓ，３Ｈ）１．４７－１．７０（ｍ，７Ｈ）１．７３（ｓ，３Ｈ）１．７７－１．９４（ｍ，６Ｈ）２．３４－２．５９（ｍ，５Ｈ）２．９６（ｔ，Ｊ＝５．４Ｈｚ １Ｈ）３．２９－３．４０（ｍ，１Ｈ）３．５２（ｓ，３Ｈ）３．５６－３．６６（ｍ，３Ｈ）３．７０（ｓ，３Ｈ）３．７７－３．８９（ｍ，１Ｈ）４．６１（ｔｄ，Ｊ＝８．０５，４．８８Ｈｚ，１Ｈ）５．４６（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８９（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２３（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）７．１０（ｄ，Ｊ＝８．２９Ｈｚ，１Ｈ）． Step 3: Methyl (S) -5-hydroxy-2-(2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-(((2R, 3R) -3-((2R, 3R) -7- 2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro -2H-pyran-2-yl) acetamide) pentanoate

Trimethylsilyldiazomethane (2.0 M, 0.095 mL, 0.19 mmol in hexanes) to a mixture of compound (35 mg, 0.063 mmol) obtained from step 2 in THF (2 mL) and methanol (0.5 mL) at 0 ° C. Was added. The mixture was then warmed to room temperature and stirred for 1 hour. The mixture was subsequently cooled to 0 ° C. and quenched by the addition of acetic acid. The resulting mixture was stirred for 30 minutes and then vacuum concentrated. The obtained residue was purified by silica gel chromatography (12 g, heptane / AcOEt = 90/10 to 30/70) to give the title compound (15.7 mg, 44% yield).
¹ H NMR (400 MHz, chloroform-d) δ ppm 0.66 (d, J = 6.8 Hz, 3H) 0.85 (d, J = 6.8 Hz, 3H) 1.03 (d, J = 6. 3Hz, 3H) 1.16 (d, J = 6.8Hz, 3H) 1.19-1.23 (m, 3H) 1.27 (s, 3H) 1.47-1.70 (m, 7H) 1.73 (s, 3H) 1.77-1.94 (m, 6H) 2.34-2.59 (m, 5H) 2.96 (t, J = 5.4Hz 1H) 3.29-3 .40 (m, 1H) 3.52 (s, 3H) 3.56-3.66 (m, 3H) 3.70 (s, 3H) 3.77-3.89 (m, 1H) 4.61 (Td, J = 8.05, 4.88Hz, 1H) 5.46 (dd, J = 15.12, 8.78Hz, 1H) 5.89 (d, J = 10.73Hz, 1H) 6.23 (Dd, J = 15.12, 10.73Hz, 1H) 7.10 (d, J = 8.29Hz, 1H).

室温でＤＣＭ（１ｍＬ）中のステップ３で生成された化合物（１５．７ｍｇ、０．０２８ｍｍｏｌ）と４－クロロギ酸ニトロフェニル（１１．１５ｍｇ、０．０５５ｍｍｏｌ）とヒューニッヒ塩基（０．０２４ｍＬ、０．１３８ｍｍｏｌ）との混合物にＤＭＡＰ（１．６８９ｍｇ、０．０１４ｍｍｏｌ）を加えた。得られた混合物を室温で１６時間撹拌した。続いて、ピペラジン（２３．８２ｍｇ、０．２７７ｍｍｏｌ）を加え、混合物を更に１時間撹拌した。次に、この混合物をＤＣＭで希釈し、有機層を分離し、水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。粗製残渣を次のステップで精製なしに使用した。 Step 4: (S) -4- (2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) ) -4-Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2-H-pyran- 2-yl) acetamide) -5-methoxy-5-oxopentylpiperazin-1-carboxylate

The compound (15.7 mg, 0.028 mmol) produced in step 3 in DCM (1 mL) at room temperature, nitrophenyl 4-chloroformate (11.15 mg, 0.055 mmol) and the Hunig base (0.024 mL, 0. DMAP (1.689 mg, 0.014 mmol) was added to the mixture with (138 mmol). The resulting mixture was stirred at room temperature for 16 hours. Subsequently, piperazine (23.82 mg, 0.277 mmol) was added and the mixture was stirred for an additional hour. The mixture was then diluted with DCM, the organic layer was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The crude residue was used in the next step without purification.

ステップ４で得られた残渣（１５ｍｇ、０．０２２ｍｍｏｌ）とＭｅＯＨ（１ｍＬ）との混合物に水酸化ナトリウム水溶液（２Ｎ、１００μｌ、０．２０ｍｍｏｌ）を加えた。この混合物を４０℃に加温し、２時間撹拌した。次に混合物を０℃に冷却し、塩酸水溶液（１００μＬ、２Ｎ）を加えた。次に得られた混合物を真空濃縮し、得られた残渣を分取ＨＰＬＣ（Ｈ_２Ｏ／ＭｅＣＮ／ＮＨ_３水溶液＝６０／４０／０．１～３０／７０／０．１）により精製して、表題化合物を無色の油として得た（７．０ｍｇ、ステップ４及び５で４８％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６６６．９０［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８１（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）０．９８－１．０４（ｍ，３Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１７（ｄ，Ｊ＝１１．７１Ｈｚ，１Ｈ）１．２２－１．２８（ｍ，４Ｈ）１．３５（ｄ，Ｊ＝６．８３Ｈｚ，１Ｈ）１．４６－１．６６（ｍ，１０Ｈ）１．７０（ｓ ３Ｈ）１．８２－１．９４（ｍ，３Ｈ）２．２５－２．３１（ｍ，１Ｈ）２．３６－２．４５（ｍ，２Ｈ）２．６１－２．６５（ｍ，１Ｈ）２．９５（ｄｄ，Ｊ＝６．１０，４．１５Ｈｚ，１Ｈ）３．１４（ｂｒ．ｓ．，２Ｈ）３．５０（ｓ，３Ｈ）３．６２－３．７１（ｍ，４Ｈ）３．７６（ｔ，Ｊ＝６．３Ｈｚ，１Ｈ）４．０３（ｄｄ，Ｊ＝４．１５，１．７１Ｈｚ，１Ｈ）４．２９（ｂｒ．ｓ．，１Ｈ）５．４５（ｄ，Ｊ＝６．３４Ｈｚ，１Ｈ）５．８７（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２７（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． Step 5: (S) -2-(2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) ) -4-Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2-H-pyran- 2-yl) acetamide) -5-((piperazine-1-carbonyl) oxy) pentanic acid (H4)

Aqueous sodium hydroxide solution (2N, 100 μl, 0.20 mmol) was added to the mixture of the residue (15 mg, 0.022 mmol) obtained in step 4 and MeOH (1 mL). The mixture was heated to 40 ° C. and stirred for 2 hours. The mixture was then cooled to 0 ° C. and aqueous hydrochloric acid solution (100 μL, 2N) was added. Next, the obtained mixture was concentrated in vacuum, and the obtained residue was purified by preparative HPLC (H2 O / MeCN / NH ₃ aqueous solution = _60/40 / 0.1 to 30/70 / 0.1). , The title compound was obtained as a colorless oil (7.0 mg, 48% yield in steps 4 and 5). LC / MS (ESI, m / z), 666.90 [M + H] ⁺ .
¹ H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.65 (d, J = 6.83 Hz, 3H) 0.81 (d, J = 7.32 Hz, 3H) 0.98-1.04 (m) , 3H) 1.08 (d, J = 6.34Hz, 3H) 1.17 (d, J = 11.71Hz, 1H) 1.22-1.28 (m, 4H) 1.35 (d, J) = 6.83Hz, 1H) 1.46-1.66 (m, 10H) 1.70 (s 3H) 1.82-1.94 (m, 3H) 2.25-2-31 (m, 1H) 2.36-2.45 (m, 2H) 2.61-2.65 (m, 1H) 2.95 (dd, J = 6.10, 4.15Hz, 1H) 3.14 (br.s. , 2H) 3.50 (s, 3H) 3.62-3.71 (m, 4H) 3.76 (t, J = 6.3Hz, 1H) 4.03 (dd, J = 4.15, 1) .71Hz, 1H) 4.29 (br.s., 1H) 5.45 (d, J = 6.34Hz, 1H) 5.87 (d, J = 10.73Hz, 1H) 6.27 (dd, dd, J = 15.12, 10.73Hz, 1H).

ＤＭＦ（０．０２８Ｍ）中のペイロード（例えば、前述のステップにより合成された化合物；１．０当量）と４－（（Ｓ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル（４－ニトロフェニル）カーボネート（１．０当量）との混合物に室温でＤＩＰＥＡ（３．０３当量）を加えた。得られた混合物を室温で１６時間撹拌し、真空濃縮し、得られた残渣を逆相分取ＨＰＬＣ（Ｈ_２Ｏ／ＭｅＣＮ／ＨＣＯＯＨ＝６０／４０／０．１～４０／６０／０．１）により精製して、所望の化合物を得た。 1.2.7 General Procedure for Synthesizing the MC-Val-Cit-pABC Linker-Payload Scheme 3 outlines the general procedure for synthesizing the MC-Val-Cit-pABC Linker-Payload.

The payload in DMF (0.028M) (eg, the compound synthesized by the steps described above; 1.0 eq) and 4-((S) -2-((S) -2- (6- (2,5)). -Dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methylbutaneamide) -5-ureidopentaneamide) With benzyl (4-nitrophenyl) carbonate (1.0 equivalent) DIPEA (3.03 eq) was added to the mixture at room temperature. The resulting mixture was stirred at room temperature for 16 hours, concentrated in vacuo and the resulting residue was subjected to reverse phase preparative HPLC ( _H2O / MeCN / HCOOH = 60/40 / 0.1-40 / 60 / 0.1). ) To obtain the desired compound.

セクション１．２．７に概説される一般的手順のとおりＡＤＬ１－Ｈ１（２－（（２Ｒ，４Ｒ，５Ｓ，６Ｓ）－４－（（４－（（（４－（（Ｒ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）オキシ）カルボニル）ピペラジン－１－カルボニル）オキシ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）酢酸）を合成し、無色の油として得た（１２．７ｍｇ、３９％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．７１（ｄ，Ｊ＝５．８５Ｈｚ，４Ｈ）０．８０（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．９４（ｄｄ，Ｊ＝ ６．１，３．２Ｈｚ，６Ｈ）１．０１（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．６Ｈｚ，３Ｈ）１．１３－１．１９（ｍ，１Ｈ）１．２５（ｓ，４Ｈ）１．２９－１．３６（ｍ，２Ｈ）１．４６－１．６５（ｍ，７Ｈ）１．６８（ｓ，３Ｈ）１．７１－１．７６（ｍ，１Ｈ）１．８４－１．９３（ｍ，２Ｈ）２．０２－２．１５（ｍ，２Ｈ）２．２５（ｔ，Ｊ＝７．３２Ｈｚ，２Ｈ）２．３７－２．５１（ｍ，３Ｈ）２．５８－２．６７（ｍ，２Ｈ）２．７９（ｓ，４Ｈ）２．８２－２．９６（ｍ，２Ｈ）３．０８－３．２３（ｍ，８Ｈ）３．３９－３．５４（ｍ，１５Ｈ）３．７６（ｔ，Ｊ＝６．３４Ｈｚ，１Ｈ）３．８５－３．９７（ｍ，２Ｈ）４．１３（ｄ，Ｊ＝７．３２Ｈｚ，１Ｈ）４．４６－４．５８（ｍ，２Ｈ）５．０７（ｓ，２Ｈ）５．４８（ｄｄ，Ｊ＝１４．８８，９．０３Ｈｚ，１Ｈ）５．９３（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）７．３０（ｄ，Ｊ＝７．８１Ｈｚ，２Ｈ）７．５７（ｄ，Ｊ＝７．８１Ｈｚ，２Ｈ）． 1.2.8 Synthesis of ADL1-H1

ADL1-H1 (2-((2R, 4R, 5S, 6S) -4-((4-(((4- ((R) -2-)- ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methylbutaneamide) -5-ureidopentaneamide) benzyl) Oxy) carbonyl) piperazin-1-carbonyl) oxy) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-) Synthesis of methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) acetic acid) And obtained as a colorless oil (12.7 mg, 39% yield).
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.71 (d, J = 5.85 Hz, 4H) 0.80 (d, J = 6.34 Hz, 3H) 0.94 (dd, J = 6) .1, 3.2Hz, 6H) 1.01 (d, J = 6.3Hz, 3H) 1.08 (d, J = 6.6Hz, 3H) 1.13-1.19 (m, 1H) 1 .25 (s, 4H) 1.29-1.36 (m, 2H) 1.46-1.65 (m, 7H) 1.68 (s, 3H) 1.71-1.76 (m, 1H) ) 1.84-1.93 (m, 2H) 2.02.2.15 (m, 2H) 2.25 (t, J = 7.32Hz, 2H) 2.37-1.51 (m, 3H) ) 2.58-2.67 (m, 2H) 2.79 (s, 4H) 2.82-2.96 (m, 2H) 3.08-3.23 (m, 8H) 3.39-3 .54 (m, 15H) 3.76 (t, J = 6.34Hz, 1H) 3.85-3.97 (m, 2H) 4.13 (d, J = 7.32Hz, 1H) 4.46 -4.58 (m, 2H) 5.07 (s, 2H) 5.48 (dd, J = 14.88, 9.03Hz, 1H) 5.93 (d, J = 10.73Hz, 1H) 6 .28 (dd, J = 14.88, 10.98Hz, 1H) 7.30 (d, J = 7.81Hz, 2H) 7.57 (d, J = 7.81Hz, 2H).

セクション１．２．７に概説される一般的手順のとおりＡＤＬ１－Ｈ４（（Ｓ）－５－（（４－（（（４－（（Ｓ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）オキシ）カルボニル）ピペラジン－１－カルボニル）オキシ）－２－（２－（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）アセトアミド）ペンタン酸）を合成し、無色の油として得た（２．４ｍｇ、４４％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６４（ｄ，Ｊ＝６．５０Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８０Ｈｚ，３Ｈ）０．８４－０．８９（ｍ，１Ｈ）０．８９－０．９７（ｍ，６Ｈ）０．９７－１．０３（ｍ，２Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１０－１．１６（ｍ，１Ｈ）１．２２－１．２５（ｍ，３Ｈ）１．２５－１．３２（ｍ，６Ｈ）１．４９－１．６６（ｍ，９Ｈ）１．６９（ｓ，３Ｈ）１．８０－１．９５（ｍ，３Ｈ）１．９５－２．１５（ｍ，２Ｈ）２．２５（ｔ，Ｊ＝７．２０Ｈｚ，２Ｈ）２．３３－２．４８（ｍ，１Ｈ）２．６２（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）２．９１－２．９８（ｍ，１Ｈ）３．０４－３．１３（ｍ，１Ｈ）３．３９－３．４７（ｍ，７Ｈ）３．４９（ｓ，３Ｈ）３．６３（ｓ，１Ｈ）３．７６（ｔ，Ｊ＝６．５０Ｈｚ，１Ｈ）３．９９（ｂｒ．ｓ，１Ｈ）４．１４（ｄ，Ｊ＝７．２０Ｈｚ，１Ｈ）４．４４－４．５１（ｍ，１Ｈ）４．４４－４．５１（ｍ，１Ｈ）５．０７（ｂｒ．ｓ，２Ｈ）５．４３（ｄｄ，Ｊ＝１５．３７，９．０３Ｈｚ，１Ｈ）５．８２－５．９０（ｍ，１Ｈ）６．１９－６．３６（ｍ，１Ｈ）６．７７（ｓ，１Ｈ）７．３０（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）７．５７（ｄ，Ｊ＝８．７８Ｈｚ，２Ｈ）． 1.2.9 ADL1-H4

ADL1-H4 ((S) -5-((4-(((4-((S) -2-((S) -2- (6) -(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methylbutaneamide) -5-ureidopentaneamide) benzyl) oxy) carbonyl) piperazin-1-carbonyl ) Oxy) -2-(2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4) -Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2-yl ) Acetamide) pentanic acid) was synthesized and obtained as a colorless oil (2.4 mg, 44% yield).
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.64 (d, J = 6.50 Hz, 3H) 0.80 (d, J = 6.80 Hz, 3H) 0.84-0.89 (m) , 1H) 0.89-0.97 (m, 6H) 0.97-1.03 (m, 2H) 1.08 (d, J = 6.34Hz, 3H) 1.10-1.16 (m) , 1H) 1.22-1.25 (m, 3H) 1.25-1.32 (m, 6H) 1.49-1.66 (m, 9H) 1.69 (s, 3H) 1.80 -1.95 (m, 3H) 1.95-2.15 (m, 2H) 2.25 (t, J = 7.20Hz, 2H) 2.33-2.48 (m, 1H) 2.62 (D, J = 9.76Hz, 1H) 2.91-2.98 (m, 1H) 3.04-3.13 (m, 1H) 3.39-3.47 (m, 7H) 3.49 (S, 3H) 3.63 (s, 1H) 3.76 (t, J = 6.50Hz, 1H) 3.99 (br.s, 1H) 4.14 (d, J = 7.20Hz, 1H) ) 4.44-4.51 (m, 1H) 4.44-4.51 (m, 1H) 5.07 (br.s, 2H) 5.43 (dd, J = 15.37, 9.03Hz) , 1H) 5.82-5.90 (m, 1H) 6.19-6.36 (m, 1H) 6.77 (s, 1H) 7.30 (d, J = 8.29Hz, 2H) 7 .57 (d, J = 8.78Hz, 2H).

セクション１．２．７に概説される一般的手順のとおりＡＤＬ１－Ｈ２（２－（（２Ｒ，４Ｒ，５Ｓ，６Ｓ）－４－（（４－（（（４－（（Ｒ）－２－（（Ｒ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）オキシ）カルボニル）－１，４－ジアゼパン－１－カルボニル）オキシ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）酢酸）を合成し、無色の油として得た（１０．４ｍｇ、３４％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６１－０．６８（ｍ，３Ｈ）０．７９（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９４（ｔ，Ｊ＝６．１０Ｈｚ，６Ｈ）１．０２（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０４－１．１０（ｍ，３Ｈ）１．１０－１．１７（ｍ，１Ｈ）１．１９－１．３１（ｍ，８Ｈ）１．４２－１．４９（ｍ，２Ｈ）１．５１－１．６５（ｍ，８Ｈ）１．６７（ｓ，３Ｈ）１．６９－１．７８（ｂｒ．ｓ，３Ｈ）１．８３－１．９３（ｍ，２Ｈ）１．９４（ｓ，１Ｈ）２．００－２．１０（ｍ，２Ｈ）２．２５（ｔ，Ｊ＝７．３２Ｈｚ，２Ｈ）２．３０－２．３８（ｍ，１Ｈ）２．３８－２．４９（ｍ，２Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．９４（ｔ，Ｊ＝５．１２Ｈｚ，１Ｈ）３．０５－３．２１（ｍ，２Ｈ）３．３９－３．４８（ｍ，７Ｈ）３．４９（ｓ，３Ｈ）３．５２－３．６３（ｍ，５Ｈ）３．６７－３．８１（ｍ，２Ｈ）３．９０（ｂｒ．ｓ．，１Ｈ）４．１７（ｔ，Ｊ＝７．３０Ｈｚ，１Ｈ）４．４５－４．５５（ｍ，２Ｈ）５．００－５．０８（ｍ，２Ｈ）５．４８（ｄｄ，Ｊ＝１４．８８，９．５１Ｈｚ，１Ｈ）５．８７－５．９５（ｍ，１Ｈ）６．２３－６．３２（ｍ，１Ｈ）６．７６（ｓ，Ｊ＝４．０１Ｈｚ，２Ｈ）７．２８（ｑ，Ｊ＝７．８１Ｈｚ，２Ｈ）７．５８（ｔ，Ｊ＝７．０７Ｈｚ，２Ｈ）． 1.2.10 ADL1-H2

ADL1-H2 (2-((2R, 4R, 5S, 6S) -4-((4-(((4- ((R) -2-)- ((R) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methylbutaneamide) -5-ureidopentaneamide) benzyl) Oxy) carbonyl) -1,4-diazepan-1-carbonyl) oxy) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4) -Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2-yl ) Acetic acid) was synthesized and obtained as a colorless oil (10.4 mg, 34% yield).
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.61-0.68 (m, 3H) 0.79 (d, J = 6.83 Hz, 3H) 0.94 (t, J = 6.10 Hz) , 6H) 1.02 (d, J = 6.34Hz, 3H) 1.04-1.10 (m, 3H) 1.10-1.17 (m, 1H) 1.19-1.31 (m) , 8H) 1.42-1.49 (m, 2H) 1.51-1.65 (m, 8H) 1.67 (s, 3H) 1.69-1.78 (br.s, 3H) 1 .83-1.93 (m, 2H) 1.94 (s, 1H) 2.00-2.10 (m, 2H) 2.25 (t, J = 7.32Hz, 2H) 2.30-2 .38 (m, 1H) 2.38-2.49 (m, 2H) 2.63 (d, J = 9.27Hz, 1H) 2.94 (t, J = 5.12Hz, 1H) 3.05 -3.21 (m, 2H) 3.39-3.48 (m, 7H) 3.49 (s, 3H) 3.52-3.63 (m, 5H) 3.67-3.81 (m) , 2H) 3.90 (br.s., 1H) 4.17 (t, J = 7.30Hz, 1H) 4.45-4.55 (m, 2H) 5.00-5.08 (m, 2H) 5.48 (dd, J = 14.88, 9.51Hz, 1H) 5.87-5.95 (m, 1H) 6.23-6.32 (m, 1H) 6.76 (s,, J = 4.01Hz, 2H) 7.28 (q, J = 7.81Hz, 2H) 7.58 (t, J = 7.07Hz, 2H).

セクション１．２．７に概説される一般的手順のとおりＡＤＬ１－Ｈ３（２－（（２Ｒ，４Ｒ，５Ｓ，６Ｓ）－４－（（３－（（（（４－（（Ｒ）－２－（（Ｒ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）オキシ）カルボニル）（メチル）アミノ）ピロリジン－１－カルボニル）オキシ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）酢酸）を合成し、無色の油として得た（１８．８ｍｇ、３７％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４） δ ｐｐｍ ０．７２（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９０－０．９７（ｍ，６Ｈ）１．０２（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１６（ｄｄ，Ｊ＝１２．９３，１１．４６Ｈｚ，１Ｈ）１．２２－１．２８（ｍ，４Ｈ）１．２８－１．３７（ｍ，２Ｈ）１．４４－１．６７（ｍ，８Ｈ）１．６８（ｓ，３Ｈ）１．９０（ｄｄ，Ｊ＝１３．４２，４．１５Ｈｚ，２Ｈ）２．００－２．１５（ｍ，４Ｈ）２．２５（ｔ，Ｊ＝７．３２Ｈｚ，２Ｈ）２．３８－２．５２（ｍ，３Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．８４（ｓ，３Ｈ）２．９３－２．９７（ｍ，１Ｈ）３．０８（ｄ，Ｊ＝６．３４Ｈｚ，１Ｈ）３．１６（ｄ，Ｊ＝６．８３Ｈｚ，１Ｈ）３．４２－３．４７（ｍ，３Ｈ）３．４９（ｓ，３Ｈ）３．５１－３．５７（ｍ，２Ｈ）３．７６（ｔ，Ｊ＝６．３４Ｈｚ，１Ｈ）３．８５－３．９２（ｍ，１Ｈ）４．１４－４．１９（ｍ，１Ｈ）４．４７－４．５５（ｍ，２Ｈ）４．７１（ｄ，Ｊ＝７．３２Ｈｚ，１Ｈ）５．０６（ｓ，２Ｈ）５．４８（ｄｄ，Ｊ＝１４．８８，９．０３Ｈｚ，１Ｈ）５．９３（ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．７６（ｓ，Ｊ＝５．１３Ｈｚ，２Ｈ）７．３０（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）７．５６（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）． 1.2.11 ADL1-H3

ADL1-H3 (2-((2R, 4R, 5S, 6S) -4-((3-((((4- ((R) -2) -2) -((R) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methylbutaneamide) -5-ureidopentaneamide) benzyl ) Oxy) carbonyl) (methyl) amino) pyrrolidine-1-carbonyl) oxy) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R)-) 4-Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2- (Il) acetic acid) was synthesized and obtained as a colorless oil (18.8 mg, 37% yield).
¹ H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.72 (d, J = 6.83 Hz, 3H) 0.80 (d, J = 6.83 Hz, 3H) 0.90-0.97 (m) , 6H) 1.02 (d, J = 6.34Hz, 3H) 1.08 (d, J = 6.34Hz, 3H) 1.16 (dd, J = 12.93, 11.46Hz, 1H) 1 .22-1.28 (m, 4H) 1.28-1.37 (m, 2H) 1.44-1.67 (m, 8H) 1.68 (s, 3H) 1.90 (dd, J) = 13.42, 4.15Hz, 2H) 2.00-2.15 (m, 4H) 2.25 (t, J = 7.32Hz, 2H) 2.38-2.52 (m, 3H) 2 .63 (d, J = 9.27Hz, 1H) 2.84 (s, 3H) 2.93-2.97 (m, 1H) 3.08 (d, J = 6.34Hz, 1H) 3.16 (D, J = 6.83Hz, 1H) 3.42-3.47 (m, 3H) 3.49 (s, 3H) 3.51-3.57 (m, 2H) 3.76 (t, J) = 6.34Hz, 1H) 3.85-3.92 (m, 1H) 4.14-4.19 (m, 1H) 4.47-4.55 (m, 2H) 4.71 (d, J) = 7.32Hz, 1H) 5.06 (s, 2H) 5.48 (dd, J = 14.88, 9.03Hz, 1H) 5.93 (d, J = 11.22Hz, 1H) 6.28 (Dd, J = 14.88, 10.98Hz, 1H) 6.76 (s, J = 5.13Hz, 2H) 7.30 (d, J = 8.29Hz, 2H) 7.56 (d, J) = 8.29Hz, 2H).

ステップ１：２－（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）アセトアミド

ＴＨＦ（１５ｍＬ）中の２－（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）酢酸（ハーボキシジエン、７５０ｍｇ、１．７１ｍｍｏｌ）とトリエチルアミン（１．１９２ｍＬ、８．５５ｍｍｏｌ）との混合物に０℃でクロロギ酸エチル（０．７６７ｍＬ、５．１３ｍｍｏｌ）を加えた。得られた混合物を０℃で３０分間撹拌し、次にメタノール中のアンモニア（７Ｍ、３．６６ｍＬ、２５．６５ｍｍｏｌ）を加えた。得られた混合物を同じ温度で更に３０分間撹拌した後、真空濃縮し、得られた残渣をシリカゲルクロマトグラフィー（ヘプタン／ＡｃＯＥｔ＝５０／５０～０／１００、次にＡｃＯＥｔ＝８０／２０）により精製して、表題化合物を無色の固体として得た（６３２ｍｇ、８５％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６７（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．７Ｈｚ， ３Ｈ）１．０５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．７Ｈｚ，３Ｈ）１．２０－１．２５（ｍ，３Ｈ）１．２７（ｓ，３Ｈ）１．３４－１．４５（ｍ，１Ｈ）１．４９－１．６５（ｍ，７Ｈ）１．７１（ｓ，３Ｈ）１．８２－１．９７（ｍ，２Ｈ）２．０１－２．０５（ｍ，１Ｈ）２．３３－２．４８（ｍ，３Ｈ）２．５４（ｄ，Ｊ＝９．２７Ｈｚ，２Ｈ）２．９５－２．９９（ｍ，１Ｈ）３．３４（ｄ，Ｊ＝１０．０Ｈｚ，１Ｈ）３．５２（ｓ，３Ｈ）３．６３－３．７０（ｍ，１Ｈ）３．８０－３．８７（ｍ，１Ｈ）４．０８－４．１４（ｍ，１Ｈ）５．２４－５．３３（ｍ，１Ｈ）５．４７（ｄｄ，Ｊ＝１５．１２，９．２７Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２２（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．６１（ｂｒ．ｓ，１Ｈ）． 1.3 Preparation of ADL1-H5, ADL1-H6, and ADL1-H7 1.3.1 Overview-General procedure 2

Step 1: 2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3) -Methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) acetamide

2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-) in THF (15 mL)) Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) Ethyl chloroformate (0.767 mL, 5.13 mmol) was added to a mixture of acetic acid (harboxydiene, 750 mg, 1.71 mmol) and triethylamine (1.192 mL, 8.55 mmol) at 0 ° C. The resulting mixture was stirred at 0 ° C. for 30 minutes and then ammonia in methanol (7M, 3.66 mL, 25.65 mmol) was added. The resulting mixture was stirred at the same temperature for another 30 minutes, then concentrated in vacuum and the resulting residue was purified by silica gel chromatography (heptane / AcOEt = 50/50 to 0/100, then AcOEt = 80/20). The title compound was obtained as a colorless solid (632 mg, 85% yield).
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.67 (d, J = 6.34 Hz, 3H) 0.85 (d, J = 6.7 Hz, 3H) 1.05 (d, J = 6. 34Hz, 3H) 1.16 (d, J = 6.7Hz, 3H) 1.20-1.25 (m, 3H) 1.27 (s, 3H) 1.34-1.45 (m, 1H) 1.49-1.65 (m, 7H) 1.71 (s, 3H) 1.82-1.97 (m, 2H) 2.01-2.05 (m, 1H) 2.33-2. 48 (m, 3H) 2.54 (d, J = 9.27Hz, 2H) 2.95-2.99 (m, 1H) 3.34 (d, J = 10.0Hz, 1H) 3.52 ( s, 3H) 3.63-3.70 (m, 1H) 3.80-3.87 (m, 1H) 4.08-4.14 (m, 1H) 5.24-5.33 (m, 1H) 1H) 5.47 (dd, J = 15.12, 9.27Hz, 1H) 5.90 (d, J = 10.73Hz, 1H) 6.22 (dd, J = 14.88, 10.98Hz, 1H) 6.61 (br.s, 1H).

０℃でＤＣＭ（１５ｍＬ）中のステップ１から分離された化合物（７１９ｍｇ、１．４４６ｍｍｏｌ）とトリエチルアミン（２．０１５ｍＬ、１４．４５８ｍｍｏｌ）との混合物に、クロロトリエチルシラン（１０９０ｍｇ、７．２２９ｍｍｏｌ）及びＮ，Ｎ－ジメチルピリジン－４－アミン（１７７ｍｇ、１．４４６ｍｍｏｌ）を加えた。次に得られた混合物を室温に加温し、１６時間撹拌した。撹拌後、この混合物をＤＣＭ（１００ｍＬ）で希釈し、混合物を水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。得られた残渣をＡｃＯＥｔ（１００ｍＬ）及びアミノ官能化シリカ（５０ｇ）と合わせた。得られた懸濁液を室温で１６時間撹拌し、次にろ過した。ろ液をＡｃＯＥｔ／ＭｅＯＨ（９／１、１５０ｍＬ）で洗浄した。合わせた母液を真空濃縮し、分離された残渣をシリカゲルクロマトグラフィー（４０ｇ、ヘプタン／ＡｃＯＥｔ＝７０／３０～０／１００）により精製して、表題化合物を無色の固体として得た（８０７ｍｇ、定量的収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６０（ｑ，Ｊ＝７．８Ｈｚ，６Ｈ）０．６７（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）０．７６（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）０．９５（ｔ，Ｊ＝７．８Ｈｚ，９Ｈ）１．０４（ｔｄ，Ｊ＝３．３，１．７Ｈｚ，６Ｈ）１．１７－１．２０（ｍ，１Ｈ）１．２４（ｓ，３Ｈ）１．３４－１．４６（ｍ，２Ｈ）１．５０－１．５８（ｍ，１Ｈ）１．６１（ｓ，３Ｈ）１．７０（ｓ，３Ｈ）１．８２－１．９２（ｍ，２Ｈ）２．３３－２．４５（ｍ，３Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）３．０５－３．０９（ｍ，１Ｈ），３．３４（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．５０（ｓ，３Ｈ）３．６２－３．７０（ｍ，１Ｈ）３．８５（ｔ，Ｊ＝６．５９Ｈｚ，１Ｈ）５．２９（ｂｒ．ｓ．，１Ｈ）５．４８（ｄｄ，Ｊ＝１４．８８，８．５４Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２１（ｄｄ，Ｊ＝１４．６４，１１．２２Ｈｚ，１Ｈ）６．６３（ｂｒ．ｓ．，１Ｈ）． Step 2: 2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -3-methoxy-4) -((Triethylsilyl) oxy) pentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2 -Il) Acetamide

Chlorotriethylsilane (1090 mg, 7.229 mmol) and chlorotriethylsilane (1090 mg, 7.229 mmol) in a mixture of compound (719 mg, 1.446 mmol) separated from step 1 in DCM (15 mL) at 0 ° C. and triethylamine (2.015 mL, 14.458 mmol). N, N-dimethylpyridin-4-amine (177 mg, 1.446 mmol) was added. The resulting mixture was then warmed to room temperature and stirred for 16 hours. After stirring, the mixture was diluted with DCM (100 mL), the mixture was washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was combined with AcOEt (100 mL) and amino-functionalized silica (50 g). The resulting suspension was stirred at room temperature for 16 hours and then filtered. The filtrate was washed with AcOEt / MeOH (9/1, 150 mL). The combined mother liquor was concentrated in vacuo and the separated residue was purified by silica gel chromatography (40 g, heptane / AcOEt = 70/30 to 0/100) to give the title compound as a colorless solid (807 mg, quantitative). yield).
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.60 (q, J = 7.8 Hz, 6H) 0.67 (d, J = 6.3 Hz, 3H) 0.76 (d, J = 7. 32Hz, 3H) 0.95 (t, J = 7.8Hz, 9H) 1.04 (td, J = 3.3, 1.7Hz, 6H) 1.17-1.20 (m, 1H) 1. 24 (s, 3H) 1.34-1.46 (m, 2H) 1.50-1.58 (m, 1H) 1.61 (s, 3H) 1.70 (s, 3H) 1.82- 1.92 (m, 2H) 2.33-2.45 (m, 3H) 2.63 (d, J = 9.27Hz, 1H) 3.05-3.09 (m, 1H), 3.34 (D, J = 9.76Hz, 1H) 3.50 (s, 3H) 3.62-3.70 (m, 1H) 3.85 (t, J = 6.59Hz, 1H) 5.29 (br) .S., 1H) 5.48 (dd, J = 14.88, 8.54Hz, 1H) 5.90 (d, J = 10.73Hz, 1H) 6.21 (dd, J = 14.64, 11.22Hz, 1H) 6.63 (br.s., 1H).

アリルアルコール（２０ｍＬ）中のステップ２で分離された化合物（８０７ｍｇ、１．４６２ｍｍｏｌ）と二酢酸ヨードベンゼン（１４１３ｍｇ、４．３８７ｍｍｏｌ）との混合物を６０℃に加熱し、３時間撹拌した。次に混合物を室温に冷却し、それにＡｃＯＥｔ及び重炭酸ナトリウム水溶液（各１００ｍＬ）を加えた。有機相を分離し、水相をＡｃＯＥｔで抽出した。合わせた有機抽出物を水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。得られた残渣をシリカゲルクロマトグラフィー（４０ｇ、ヘプタン／ＡｃＯＥｔ＝９０／１０～５０／５０）により精製して、表題化合物を無色の油として得た（６６３ｍｇ、７５％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．５５－０．６７（ｍ，９Ｈ）０．７７（ｄ，Ｊ＝６．７Ｈｚ，３Ｈ）０．８７（ｔ，Ｊ＝６．５Ｈｚ，２Ｈ）０．９５（ｔ，Ｊ＝７．８Ｈｚ，９Ｈ）１．０４（ｔ，Ｊ＝５．５Ｈｚ，６Ｈ）１．１６－１．３２（ｍ，９Ｈ）１．３９－１．５３（ｍ，２Ｈ）１．５８（ｓ，３Ｈ）１．６９（ｓ，３Ｈ）１．８０－１．９１（ｍ，２Ｈ）２．４０（ｂｒ．ｓ，１Ｈ）２．６４（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）３．０１－３．１０（ｍ，２Ｈ）３．２６（ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）３．３５－３．４４（ｍ，２Ｈ）３．５０（ｓ，３Ｈ）３．８５（ｔ，Ｊ＝６．５Ｈｚ，１Ｈ）４．５４（ｄ，Ｊ＝５．３７Ｈｚ，２Ｈ）５．１２（ｂｒ．ｓ．，１Ｈ）５．１９（ｄｔ，Ｊ＝１０．４９，１．１０Ｈｚ，１Ｈ）５．２５－５．３３（ｍ，１Ｈ）５．４５（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８５－５．９７（ｍ，２Ｈ）６．２２（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）． Step 3: Allyl (((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -3-methoxy-4) -((Triethylsilyl) oxy) pentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2 -Il) Methyl) Carbamate

A mixture of the compound (807 mg, 1.462 mmol) separated in step 2 in allyl alcohol (20 mL) and iodobenzene diacetate (1413 mg, 4.387 mmol) was heated to 60 ° C. and stirred for 3 hours. The mixture was then cooled to room temperature and AcOEt and aqueous sodium bicarbonate solution (100 mL each) were added. The organic phase was separated and the aqueous phase was extracted with AcOEt. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The obtained residue was purified by silica gel chromatography (40 g, heptane / AcOEt = 90/10 to 50/50) to give the title compound as a colorless oil (663 mg, 75% yield).
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.55-0.67 (m, 9H) 0.77 (d, J = 6.7 Hz, 3H) 0.87 (t, J = 6.5 Hz, 2H) 0.95 (t, J = 7.8Hz, 9H) 1.04 (t, J = 5.5Hz, 6H) 1.16-1.32 (m, 9H) 1.39-1.53 ( m, 2H) 1.58 (s, 3H) 1.69 (s, 3H) 1.80-1.91 (m, 2H) 2.40 (br.s, 1H) 2.64 (d, J = 9.27Hz, 1H) 3.01-3.10 (m, 2H) 3.26 (d, J = 10.24Hz, 1H) 3.35-3.44 (m, 2H) 3.50 (s, 3H) 3.85 (t, J = 6.5Hz, 1H) 4.54 (d, J = 5.37Hz, 2H) 5.12 (br.s., 1H) 5.19 (dt, J = 10) .49, 1.10Hz, 1H) 5.25-5.33 (m, 1H) 5.45 (dd, J = 15.12, 8.78Hz, 1H) 5.85-5.97 (m, 2H) ) 6.22 (dd, J = 14.88, 10.98Hz, 1H).

ステップ３で得られた化合物（６６３ｍｇ、１．０９１ｍｍｏｌ）とＴＨＦ（１５ｍＬ、１８３．０６５ｍｍｏｌ）との混合物にボラン－ジメチルアミン錯体（６４３ｍｇ、１０．９０６ｍｍｏｌ）を加えた。ベッセルを窒素でパージした後、テトラキス（トリフェニルホスフィン）パラジウム（０）（３７．８ｍｇ、０．０３３ｍｍｏｌ）を加え、混合物を室温で１時間撹拌した。次にこの混合物をＡｃＯＥｔ及び飽和重炭酸ナトリウム水溶液（各１００ｍＬ）で希釈し、相を分離させた。水層をＡｃＯＥｔ（５０ｍＬ×３）で抽出し、次に合わせた有機画分を水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。得られた残渣をアミノ官能化（ａｍｉｎｏ－ｆｕｎｃｔｉｏｎａｌｉｅｄ）シリカゲルクロマトグラフィー（４０ｇ、ヘプタン／ＡｃＯＥｔ＝７０／３０～０／１００）により精製して（ｐｕｒｉｅｄ）、表題化合物を白色固体として得た（５２５ｍｇ、９２％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６０（ｑ，Ｊ＝７．８０Ｈｚ，６Ｈ）０．６５（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）０．７７（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９５（ｔ，Ｊ＝７．８Ｈｚ，９Ｈ）１．０３（ｄｄ，Ｊ＝９．０，６．６Ｈｚ，６Ｈ）１．１４－１．２１（ｍ，１Ｈ）１．２３（ｓ，３Ｈ），１．３０－１．３４（ｍ，１Ｈ）１．４２（ｄｄｄ，Ｊ＝９．４，７，２．７Ｈｚ，１Ｈ）１．４８－１．５９（ｍ，６Ｈ）１．７０（ｓ，３Ｈ）１．８１－１．９１（ｍ，２Ｈ）２．３４－２．４４（ｍ，１Ｈ）２．６２－２．７４（ｍ，３Ｈ）３．０６（ｄｄ，Ｊ＝６．８３，２．９３Ｈｚ，１Ｈ）３．２５－３．３１（ｍ，２Ｈ）３．５０（ｓ，３Ｈ）３．８１－３．８９（ｑｕｉｎ，Ｊ＝６．５Ｈｚ，１Ｈ）５．４４（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８８（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２２（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． Step 4: ((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -3-methoxy-4-( (Triethylsilyl) Oxy) Pentane-2-yl) -2-Methyloxylan-2-yl) -6-Methylhepta-2,4-diene-2-yl) -5-Methyltetrahydro-2H-pyran-2-yl ) Methylamine

A borane-dimethylamine complex (643 mg, 10.906 mmol) was added to the mixture of the compound (663 mg, 1.091 mmol) obtained in step 3 and THF (15 mL, 183.065 mmol). After purging the vessel with nitrogen, tetrakis (triphenylphosphine) palladium (0) (37.8 mg, 0.033 mmol) was added and the mixture was stirred at room temperature for 1 hour. The mixture was then diluted with AcOEt and saturated aqueous sodium bicarbonate solution (100 mL each) to separate the phases. The aqueous layer was extracted with AcOEt (50 mL x 3), then the combined organic fractions were washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by amino-functionalized silica gel chromatography (40 g, heptane / AcOEt = 70/30 to 0/100) to give the title compound as a white solid (525 mg, 92% yield).
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.60 (q, J = 7.80 Hz, 6H) 0.65 (d, J = 6.8 Hz, 3H) 0.77 (d, J = 6. 83Hz, 3H) 0.95 (t, J = 7.8Hz, 9H) 1.03 (dd, J = 9.0, 6.6Hz, 6H) 1.14-1.21 (m, 1H) 1. 23 (s, 3H), 1.30-1.34 (m, 1H) 1.42 (ddd, J = 9.4,7,2.7Hz, 1H) 1.48-1.59 (m, 6H) ) 1.70 (s, 3H) 1.81-1.91 (m, 2H) 2.34-2.44 (m, 1H) 2.62-2.74 (m, 3H) 3.06 (dd) , J = 6.83, 2.93Hz, 1H) 3.25-3.31 (m, 2H) 3.50 (s, 3H) 3.81-3.89 (quin, J = 6.5Hz, 1H) ) 5.44 (dd, J = 15.12, 8.78Hz, 1H) 5.88 (d, J = 10.73Hz, 1H) 6.22 (dd, J = 15.12, 10.73Hz, 1H) ).

ＴＨＦ（２ｍＬ）中のステップ５で分離された化合物（１３３ｍｇ、０．２５４ｍｍｏｌ）と６－メトキシ－６－オキソヘキサン酸（０．０５６ｍｌ、０．３８１ｍｍｏｌ）との混合物にＨＡＴＵ（１４５ｍｇ、０．３８１ｍｍｏｌ）を加え、次に混合物を室温で１６時間撹拌した。続いて、ＡｃＯＥｔを加え、得られた混合物を水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。得られた残渣をＴＨＦ（２ｍＬ）と合わせ、０℃に冷却した。次に、水素化ホウ素リチウム（２２．１ｍｇ、１．０１６ｍｍｏｌ）を加え、混合物を同じ温度で３０分間撹拌した。続いて、この混合物を室温に加温し、更に１時間撹拌した。この混合物に飽和塩化アンモニウム水溶液を加え、続いてＡｃＯＥｔ及び水を加えた。有機相を分離し、水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。次に得られた残渣をＴＨＦ（５ｍＬ、６１．０２ｍｍｏｌ）に溶解させて、ＴＢＡＦ（ＴＨＦ中１Ｍ溶液、０．５０８ｍＬ、０．５０８ｍｍｏｌ）を０℃で加えた。得られた混合物を０℃で３０分間撹拌し、次に室温に加温し、続いて室温で更に２時間撹拌した。次にこの混合物をＡｃＯＥｔで希釈し、有機相を分離し、水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。得られた残渣をシリカゲルクロマトグラフィー（４０ｇシリカ、０～１００％ヘプタン／ＥｔＯＡｃ）により精製して、表題化合物を無色の油として得た（１１３ｍｇ、８５％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６４（ｄ，Ｊ＝６．３Ｈｚ，３Ｈ）０．８４（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．３Ｈ，３Ｈ）１．１４（ｄ，Ｊ＝６．３Ｈ，３Ｈ）１．１７－１．２１（ｍ，３Ｈ）１．２５（ｓ，３Ｈ）１．２７－１．４０（ｍ，３Ｈ）１．４６－１．６０（ｍ，６Ｈ）１．６０－１．６７（ｍ，２Ｈ）１．６９（ｓ，３Ｈ）１．７７－１．９２（ｍ，３Ｈ）２．１５（ｔ，Ｊ＝７．３Ｈｚ，２Ｈ）２．４０（ｂｒ．ｓ，１Ｈ）２．５３（ｄ，Ｊ＝９．７６Ｈｚ，２Ｈ）２．９２－３．０２（ｍ，２Ｈ）３．２６（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．３４－３．４５（ｍ，１Ｈ）３．５０（ｓ，３Ｈ）３．５２－３．６２（ｍ，３Ｈ）３．８０－３．８３（ｍ，１Ｈ）５．４５（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８９（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）５．９６（ｂｒ．ｓ，１Ｈ）６．２３（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）． Step 5: 6-Hydroxy-N-(((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R)-)- 4-Hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2- Methyl) -N-methylhexaneamide

HATU (145 mg, 0.381 mmol) in a mixture of the compound (133 mg, 0.254 mmol) separated in step 5 in THF (2 mL) and 6-methoxy-6-oxohexanoic acid (0.056 ml, 0.381 mmol). ) Was added, and then the mixture was stirred at room temperature for 16 hours. Subsequently, AcOEt was added, the resulting mixture was washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The obtained residue was combined with THF (2 mL) and cooled to 0 ° C. Next, lithium borohydride (22.1 mg, 1.016 mmol) was added and the mixture was stirred at the same temperature for 30 minutes. Subsequently, the mixture was heated to room temperature and stirred for another hour. Saturated aqueous ammonium chloride solution was added to this mixture, followed by AcOEt and water. The organic phase was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then dissolved in THF (5 mL, 61.02 mmol) and TBAF (1 M solution in THF, 0.508 mL, 0.508 mmol) was added at 0 ° C. The resulting mixture was stirred at 0 ° C. for 30 minutes, then warmed to room temperature and then at room temperature for an additional 2 hours. The mixture was then diluted with AcOEt, the organic phase was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (40 g silica, 0-100% heptane / EtOAc) to give the title compound as a colorless oil (113 mg, 85% yield).
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.64 (d, J = 6.3 Hz, 3H) 0.84 (d, J = 6.8 Hz, 3H) 1.03 (d, J = 6. 3H, 3H) 1.14 (d, J = 6.3H, 3H) 1.17-1.21 (m, 3H) 1.25 (s, 3H) 1.27-1.40 (m, 3H) 1.46-1.60 (m, 6H) 1.60-1.67 (m, 2H) 1.69 (s, 3H) 1.77-1.92 (m, 3H) 2.15 (t, J = 7.3Hz, 2H) 2.40 (br.s, 1H) 2.53 (d, J = 9.76Hz, 2H) 2.92-3.02 (m, 2H) 3.26 (d, J = 9.76Hz, 1H) 3.34-3.45 (m, 1H) 3.50 (s, 3H) 3.52-3.62 (m, 3H) 3.80-3.83 (m, 1H) 5.45 (dd, J = 15.12, 8.78Hz, 1H) 5.89 (d, J = 10.73Hz, 1H) 5.96 (br.s, 1H) 6.23 (dd, dd, J = 14.88, 10.98Hz, 1H).

Step 6: General procedure for synthesizing carbamate Compound (28.3 mg, 0.054 mmol) separated from step 5 in DCM (0.05 M), 4-nitrophenyl chloroformate (2.0 eq) and Hunig base (2.0 eq). DMAP (0.5 eq) was added to the mixture with 5 eq). The resulting mixture was stirred at room temperature for 16 hours. Then amine (2.0 eq) was added and the mixture was stirred for an additional hour. The mixture was then diluted with dichloromethane, the organic layer was separated, washed with water and brine, dried over sodium sulphate, filtered and concentrated in vacuo. The obtained residue was then purified by amino-functionalized silica gel chromatography to give the desired compound.

６－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－６－オキソヘキシルピペラジン－１－カルボキシレート（Ｈ５）
セクション１．３．１に概説されるステップ１～６を用いて表題化合物を無色の油として得た（２４．５ｍｇ、７１％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６３６．８９［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９５（ｄｄ，Ｊ＝１６．８，６．６Ｈｚ，１Ｈ）１．０３（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１８－１．２５（ｍ，２Ｈ）１．２６（ｓ，３Ｈ）１．２９－１．４０（ｍ，３Ｈ）１．４７－１．６９（ｍ，８Ｈ）１．７０（ｓ，３Ｈ）１．７９－１．９２（ｍ，２Ｈ）１．９２－２．０７（ｍ，４Ｈ）２．１５（ｔ，Ｊ＝７．５６Ｈｚ，２Ｈ）２．３６－２．４５（ｍ，１Ｈ）２．５４（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．８０（ｂｒ．ｓ．，４Ｈ）２．８９－３．０４（ｍ，２Ｈ）３．２７（ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）３．３５－３．４６（ｍ，５Ｈ）３．５１（ｓ，３Ｈ）３．５２－３．５８（ｍ，２Ｈ）３．８３（ｔ，Ｊ＝６．３４Ｈｚ，１Ｈ）４．０５（ｔ，Ｊ＝６．３４Ｈｚ，２Ｈ）５．４６（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８２－５．９５（ｍ，２Ｈ）６．２４（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． 1.3.2 Synthesis of ADL1-H5 1.3.2.1 Synthesis of H5

6-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-) Methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl) amino) -6-oxohexylpiperazin-1-carboxylate (H5)
Steps 1-6 outlined in Section 1.3.1 were used to give the title compound as a colorless oil (24.5 mg, 71% yield). LC / MS (ESI, m / z), 636.89 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.34 Hz, 3H) 0.85 (d, J = 6.83 Hz, 3H) 0.95 (dd, J = 16. 8,6.6Hz, 1H) 1.03 (d, J = 6.34Hz, 3H) 1.15 (d, J = 6.34Hz, 3H) 1.18-1.25 (m, 2H) 1. 26 (s, 3H) 1.29-1.40 (m, 3H) 1.47-1.69 (m, 8H) 1.70 (s, 3H) 1.79-1.92 (m, 2H) 1.92-2.07 (m, 4H) 2.15 (t, J = 7.56Hz, 2H) 2.36-2.45 (m, 1H) 2.54 (d, J = 9.27Hz, 1H) 2.80 (br.s., 4H) 2.89-3.04 (m, 2H) 3.27 (d, J = 10.24Hz, 1H) 3.35-3.46 (m, 5H) ) 3.51 (s, 3H) 3.52-3.58 (m, 2H) 3.83 (t, J = 6.34Hz, 1H) 4.05 (t, J = 6.34Hz, 2H) 5 .46 (dd, J = 15.12, 8.78Hz, 1H) 5.82-5.95 (m, 2H) 6.24 (dd, J = 15.12, 10.73Hz, 1H).

１－（４－（（Ｓ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）４－（６－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－６－オキソヘキシル）ピペラジン－１，４－ジカルボキシレート（ＡＤＬ１－Ｈ５）
セクション１．２．７に概説される手順を用いて表題化合物を無色の油として得た（２５．８ｍｇ、５４％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９４（ｄｄ，Ｊ＝６．３４，４．３９Ｈｚ，７Ｈ）１．０１（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０７（ｄ，Ｊ＝６．３０Ｈｚ，４Ｈ）１．１１－１．３８（ｍ，１４Ｈ）１．４５－１．６６（ｍ，１４Ｈ）１．６９（ｓ，３Ｈ）１．７０－１．７９（ｍ，１Ｈ）１．８０－１．９２（ｍ，３Ｈ）２．００－２．０９（ｍ，１Ｈ）２．１７（ｔ，Ｊ＝７．０７Ｈｚ，２Ｈ）２．２５（ｔ，Ｊ＝７．３２Ｈｚ，３Ｈ）２．４２（ｂｒ．ｓ．，１Ｈ）２．６３（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）２．９５（ｄｄ，Ｊ＝５．８５，４．３９Ｈｚ，１Ｈ）３．０２－３．１１（ｍ，２Ｈ）３．１７（ｄ，Ｊ＝６．８３Ｈｚ，１Ｈ）３．３７－３．４８（ｍ，１４Ｈ）３．５０（ｓ，４Ｈ）３．５３（ｔ，Ｊ＝４．５０Ｈｚ，２Ｈ）３．６３－３．６７（ｍ，２Ｈ）３．７６（ｔ，Ｊ＝６．３４Ｈｚ，１Ｈ）４．０５（ｔ，Ｊ＝６．３４Ｈｚ，３Ｈ）４．１４（ｄ，Ｊ＝７．３２Ｈｚ，１Ｈ）４．４８（ｄｄ，Ｊ＝８．７８，４．８８Ｈｚ，１Ｈ）５．０６（ｓ，２Ｈ）５．４６（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２０－６．３８（ｍ，１Ｈ）６．７６（ｓ ２Ｈ）７．３０（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）７．５７（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）． 1.3.2.2 Synthesis of ADL1-H5

1-(4-((S) -2-((S) -2-(6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methyl) Butanamide) -5-Ureidopentanamide) benzyl) 4-(6-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-) ((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5 Methyltetrahydro-2H-pyran-2-yl) methyl) amino) -6-oxohexyl) piperazin-1,4-dicarboxylate (ADL1-H5)
The title compound was obtained as a colorless oil using the procedure outlined in Section 1.2.7 (25.8 mg, 54% yield).
¹ H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.65 (d, J = 6.83 Hz, 3H) 0.80 (d, J = 6.83 Hz, 3H) 0.94 (dd, J = 6) .34, 4.39Hz, 7H) 1.01 (d, J = 6.34Hz, 3H) 1.07 (d, J = 6.30Hz, 4H) 1.11-1.38 (m, 14H) 1 .45-1.66 (m, 14H) 1.69 (s, 3H) 1.70-1.79 (m, 1H) 1.80-1.92 (m, 3H) 2.00-2.09 (M, 1H) 2.17 (t, J = 7.07Hz, 2H) 2.25 (t, J = 7.32Hz, 3H) 2.42 (br.s., 1H) 2.63 (d, J = 9.76Hz, 1H) 2.95 (dd, J = 5.85, 4.39Hz, 1H) 3.02-3.11 (m, 2H) 3.17 (d, J = 6.83Hz, 1H) 3.37-3.48 (m, 14H) 3.50 (s, 4H) 3.53 (t, J = 4.50Hz, 2H) 3.63-3.67 (m, 2H) 3. 76 (t, J = 6.34Hz, 1H) 4.05 (t, J = 6.34Hz, 3H) 4.14 (d, J = 7.32Hz, 1H) 4.48 (dd, J = 8. 78, 4.88Hz, 1H) 5.06 (s, 2H) 5.46 (dd, J = 15.12, 8.78Hz, 1H) 5.90 (d, J = 10.73Hz, 1H) 6. 20-6.38 (m, 1H) 6.76 (s 2H) 7.30 (d, J = 8.29Hz, 2H) 7.57 (d, J = 8.29Hz, 2H).

６－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－６－オキソヘキシル１，４－ジアゼパン－１－カルボキシレート（Ｈ６）
セクション１．３．１に概説されるステップ１～６を用いて表題化合物を無色の油として得た（１５．３ｍｇ、４３％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６５０．９２［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．９３－１．００（ｍ，１Ｈ）１．０３（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１８－１．２４（ｍ，２Ｈ）１．２７（ｓ，３Ｈ）１．２９－１．４１（ｍ，３Ｈ）１．４９－１．５５（ｍ，２Ｈ）１．５９－１．６８（ｍ，５Ｈ）１．７１（ｓ，３Ｈ）１．７３－１．９２（ｍ，７Ｈ）２．１６（ｔ，Ｊ＝７．５６Ｈｚ，２Ｈ）２．３６－２．４６（ｍ，１Ｈ）２．５４（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．８１－２．９３（ｍ，４Ｈ）２．９３－３．０４（ｍ，２Ｈ）３．２７（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．３７－３．４９（ｍ，４Ｈ）３．５２（ｓ，３Ｈ）３．５４－３．５９（ｍ，２Ｈ）３．７９－３．８８（ｍ，１Ｈ）４．０５（ｔ，Ｊ＝６．３４Ｈｚ，２Ｈ）５．４６（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８２－５．９６（ｍ，２Ｈ）６．２４（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． 1.3.3 Synthesis of ADL1-H6 1.3.3.1 Synthesis of H6

6-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-) Methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl) amino) -6-oxohexyl 1,4-diazepan-1-carboxylate (H6)
Steps 1-6 outlined in Section 1.3.1 were used to give the title compound as a colorless oil (15.3 mg, 43% yield). LC / MS (ESI, m / z), 650.92 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.34 Hz, 3H) 0.85 (d, J = 6.34 Hz, 3H) 0.93-1.00 (m, 1H) 1.03 (d, J = 6.83Hz, 3H) 1.16 (d, J = 6.83Hz, 3H) 1.18-1.24 (m, 2H) 1.27 (s, 3H) 1.29-1.41 (m, 3H) 1.49-1.55 (m, 2H) 1.59-1.68 (m, 5H) 1.71 (s, 3H) 1.73-1. 92 (m, 7H) 2.16 (t, J = 7.56Hz, 2H) 2.36-2.46 (m, 1H) 2.54 (d, J = 9.27Hz, 1H) 2.81- 2.93 (m, 4H) 2.93-3.04 (m, 2H) 3.27 (d, J = 9.76Hz, 1H) 3.37-3.49 (m, 4H) 3.52 ( s, 3H) 3.54-3.59 (m, 2H) 3.79-3.88 (m, 1H) 4.05 (t, J = 6.34Hz, 2H) 5.46 (dd, J = 15.12, 8.78Hz, 1H) 5.82-5.96 (m, 2H) 6.24 (dd, J = 15.12, 10.73Hz, 1H).

１－（４－（（Ｓ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）４－（６－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－６－オキソヘキシル）１，４－ジアゼパン－１，４－ジカルボキシレート（ＡＤＬ－Ｈ６）
セクション１．２．７に概説される手順を用いて表題化合物を無色の油として得た（１１．６ｍｇ、５２％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，２Ｈ）０．９４（ｄｄ，Ｊ＝６．８３，４．３９Ｈｚ，６Ｈ）０．９８－１．１６（ｍ，９Ｈ）１．１９－１．３６（ｍ，９Ｈ）１．４４－１．６５（ｍ，１４Ｈ）１．６９（ｓ，３Ｈ）１．７１－１．７８（ｍ，４Ｈ）１．８０－１．９２（ｍ，３Ｈ）２．００－２．１０（ｍ，１Ｈ）２．１１－２．１９（ｍ，２Ｈ）２．２５（ｔ，Ｊ＝７．５６Ｈｚ，３Ｈ）３．０３－３．２１（ｍ，４Ｈ）３．３６－３．４９（ｍ，１０Ｈ）３．４９－３．５１（ｍ，３Ｈ）３．５１－３．５７（ｍ，１２Ｈ）３．６２－３．６７（ｍ，８Ｈ）３．７０－３．８２（ｍ，１Ｈ）３．９７－４．０５（ｍ，２Ｈ）４．１４（ｄ，Ｊ＝７．３２Ｈｚ，１Ｈ）４．４６－４．５２（ｍ，１Ｈ）５．００－５．０８（ｍ，３Ｈ）５．３５－５．５２（ｍ，１Ｈ）５．３５－５．５２（ｍ，１Ｈ）５．８７－５．９５（ｍ，１Ｈ）６．２４－６．３３（ｍ，１Ｈ）６．７６（ｓ，２Ｈ）７．２６－７．３７（ｍ，３Ｈ）７．５７（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）８．１９－８．２４（ｍ，１Ｈ）． 1.3.3.2 Synthesis of ADL1-H6

1-(4-((S) -2-((S) -2-(6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methyl) Butanamide) -5-Ureidopentanamide) benzyl) 4-(6-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-) ((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene-2-yl) -5 Methyltetrahydro-2H-pyran-2-yl) methyl) amino) -6-oxohexyl) 1,4-diazepan-1,4-dicarboxylate (ADL-H6)
The title compound was obtained as a colorless oil using the procedure outlined in Section 1.2.7 (11.6 mg, 52% yield).
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.65 (d, J = 6.34 Hz, 3H) 0.80 (d, J = 6.83 Hz, 2H) 0.94 (dd, J = 6) .83, 4.39Hz, 6H) 0.98-1.16 (m, 9H) 1.19-1.36 (m, 9H) 1.44-1.65 (m, 14H) 1.69 (s) , 3H) 1.71-1.78 (m, 4H) 1.80-1.92 (m, 3H) 2.00-2.10 (m, 1H) 2.11-2.19 (m, 2H) ) 2.25 (t, J = 7.56Hz, 3H) 3.03-3.21 (m, 4H) 3.36-3.49 (m, 10H) 3.49-3.51 (m, 3H) ) 3.51-3.57 (m, 12H) 3.62-3.67 (m, 8H) 3.70-3.82 (m, 1H) 3.97-4.05 (m, 2H) 4 .14 (d, J = 7.32Hz, 1H) 4.46-4.52 (m, 1H) 5.00-5.08 (m, 3H) 5.35-5.52 (m, 1H) 5 .35-5.52 (m, 1H) 5.87-5.95 (m, 1H) 6.24-6.33 (m, 1H) 6.76 (s, 2H) 7.26-7.37 (M, 3H) 7.57 (d, J = 7.32Hz, 3H) 8.19-8.24 (m, 1H).

６－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－６－オキソヘキシル３－（メチルアミノ）ピロリジン－１－カルボキシレート（Ｈ７）
セクション１．３．１に概説されるステップ１～６を用いて表題化合物を無色の油として得た（２６．６ｍｇ、７６％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６５０．８８［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．９３－０．９９（ｍ，１Ｈ）１．０４（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１６（ｄｄ，Ｊ＝６．３４，０．９８Ｈｚ，３Ｈ）１．１９－１．２２（ｍ，２Ｈ）１．２７（ｓ，３Ｈ）１．３１－１．４１（ｍ，３Ｈ）１．４７－１．６７（ｍ，８Ｈ）１．７０（ｓ，４Ｈ）１．８０－１．８７（ｍ，２Ｈ）１．８９（ｄ，Ｊ＝３．９０Ｈｚ，１Ｈ）２．０３（ｄｄ，Ｊ＝１２．６８，６．３４Ｈｚ，１Ｈ）２．１５（ｔ，Ｊ＝７．３２Ｈｚ，２Ｈ）２．４１（ｍ，３Ｈ）２．５４（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）２．９４－３．１３（ｍ，２Ｈ）３．２０（ｍ，１Ｈ）３．２７（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．３５－３．５０（ｍ，３Ｈ）３．５１（ｓ，３Ｈ）３．５３－３．６０（ｍ，２Ｈ）３．７９－３．８８（ｍ，１Ｈ）４．０４（ｔ，Ｊ＝６．３４Ｈｚ，２Ｈ）５．４６（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８２－５．９６（ｍ，２Ｈ）６．２４（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）． 1.3.4 Synthesis of ADL1-H7 1.3.4.1 Synthesis of H7

6-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-) Methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl) amino) -6-oxohexyl 3- (methylamino) pyrrolidine-1-carboxylate (H7)
Steps 1-6 outlined in Section 1.3.1 were used to give the title compound as a colorless oil (26.6 mg, 76% yield). LC / MS (ESI, m / z), 650.88 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.34 Hz, 3H) 0.85 (d, J = 6.34 Hz, 3H) 0.93-0.99 (m, 1H) 1.04 (d, J = 6.34Hz, 3H) 1.16 (dd, J = 6.34, 0.98Hz, 3H) 1.19-1.22 (m, 2H) 1.27 ( s, 3H) 1.31-1.41 (m, 3H) 1.47-1.67 (m, 8H) 1.70 (s, 4H) 1.80-1.87 (m, 2H) 1. 89 (d, J = 3.90Hz, 1H) 2.03 (dd, J = 12.68, 6.34Hz, 1H) 2.15 (t, J = 7.32Hz, 2H) 2.41 (m, 3H) 2.54 (d, J = 9.76Hz, 1H) 2.94-3.13 (m, 2H) 3.20 (m, 1H) 3.27 (d, J = 9.76Hz, 1H) 3.35-3.50 (m, 3H) 3.51 (s, 3H) 3.53-3.60 (m, 2H) 3.79-3.88 (m, 1H) 4.04 (t, 1H) J = 6.34Hz, 2H) 5.46 (dd, J = 15.12, 8.78Hz, 1H) 5.82-5.96 (m, 2H) 6.24 (dd, J = 14.88, 10.98Hz, 1H).

６－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－６－オキソヘキシル３－（（（（４－（（Ｓ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）オキシ）カルボニル）（メチル）アミノ）ピロリジン－１－カルボキシレート（ＡＤＬ１－Ｈ７）
セクション１．２．７に概説される手順を用いて（混合物は２時間ではなく１６時間撹拌した）、表題化合物を無色の油として得た（２０．１ｍｇ、５４％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３０Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９４（ｄｄ，Ｊ＝６．５９，４．６３Ｈｚ，７Ｈ）１．０１（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，４Ｈ）１．１１－１．２３（ｍ，４Ｈ）１．２３－１．２７（ｍ，６Ｈ）１．２７－１．３８（ｍ，５Ｈ）１．４３－１．６５（ｍ，１４Ｈ）１．６９（ｓ，３Ｈ）１．７０－１．７７（ｍ，１Ｈ）１．８０－１．９２（ｍ，３Ｈ）２．００－２．０９（ｍ，３Ｈ）２．１７（ｔ，Ｊ＝７．３２Ｈｚ，２Ｈ）２．２５（ｔ，Ｊ＝７．３０Ｈｚ，３Ｈ）２．３８－２．４６（ｍ，１Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．８３（ｓ，３Ｈ）２．８４（ｓ，３Ｈ）２．９２－２．９６（ｍ，１Ｈ）２．９７（ｓ，３Ｈ）３．０２－３．２１（ｍ，４Ｈ）３．３０－３．４２（ｍ，３Ｈ）３．４５（ｔ，Ｊ＝７．０７Ｈｚ，３Ｈ）３．５０（ｓ，３Ｈ）３．５１－３．５８（ｍ，３Ｈ）３．７２－３．８０（ｍ，１Ｈ）４．０３（ｔ，Ｊ＝６．３４Ｈｚ，２Ｈ）４．１５（ｄ，Ｊ＝７．３２Ｈｚ，１Ｈ）４．４９（ｄｄ，Ｊ＝９．０３，５．１２Ｈｚ，１Ｈ）５．０６（ｓ，２Ｈ）５．４６（ｄｄ，Ｊ＝１４．８８，９．０３Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．７６（ｓ，２Ｈ）７．３０（ｄ，Ｊ＝５．９０Ｈｚ，２Ｈ）７．５７（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）７．９５（ｓ，１Ｈ）． 1.3.4.2 Synthesis of ADL1-H7

6-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-) Methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl) amino) -6-oxohexyl 3-((((4-((S) -2-((S) -2-(6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) ) Hexaamide) -3-methylbutaneamide) -5-ureidopentaneamide) benzyl) oxy) carbonyl) (methyl) amino) pyrrolidine-1-carboxylate (ADL1-H7)
Using the procedure outlined in Section 1.2.7 (the mixture was stirred for 16 hours instead of 2 hours), the title compound was obtained as a colorless oil (20.1 mg, 54% yield).
¹ H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.65 (d, J = 6.30 Hz, 3H) 0.80 (d, J = 6.83 Hz, 3H) 0.94 (dd, J = 6) .59, 4.63Hz, 7H) 1.01 (d, J = 6.34Hz, 3H) 1.08 (d, J = 6.34Hz, 4H) 1.11-1.23 (m, 4H) 1 .23-1.27 (m, 6H) 1.27-1.38 (m, 5H) 1.43-1.65 (m, 14H) 1.69 (s, 3H) 1.70-1.77 (M, 1H) 1.80-1.92 (m, 3H) 2.00-2.09 (m, 3H) 2.17 (t, J = 7.32Hz, 2H) 2.25 (t, J) = 7.30Hz, 3H) 2.38-2.46 (m, 1H) 2.63 (d, J = 9.27Hz, 1H) 2.83 (s, 3H) 2.84 (s, 3H) 2 .92-2.96 (m, 1H) 2.97 (s, 3H) 3.02-3.21 (m, 4H) 3.30-3.42 (m, 3H) 3.45 (t, J) = 7.07Hz, 3H) 3.50 (s, 3H) 3.51-3.58 (m, 3H) 3.72-3.80 (m, 1H) 4.03 (t, J = 6.34Hz) , 2H) 4.15 (d, J = 7.32Hz, 1H) 4.49 (dd, J = 9.03, 5.12Hz, 1H) 5.06 (s, 2H) 5.46 (dd, J) = 14.88, 9.03Hz, 1H) 5.90 (d, J = 11.22Hz, 1H) 6.28 (dd, J = 14.88, 10.98Hz, 1H) 6.76 (s, 2H) ) 7.30 (d, J = 5.90Hz, 2H) 7.57 (d, J = 8.29Hz, 2H) 7.95 (s, 1H).

ステップ１：３－ヒドロキシプロパン酸ｔｅｒｔ－ブチル

ＤＭＦ（３０ｍＬ）中の水素化ナトリウム（２６９ｍｇ、６．１５７ｍｍｏｌ）の混合物に０℃で３－ヒドロキシプロパン酸ｔｅｒｔ－ブチル（０．９０９ｍＬ、６．１５７ｍｍｏｌ）を加えた。この混合物を０℃で１時間撹拌し、次にブロモ酢酸メチル（０．６２４ｍＬ、６．１５７ｍｍｏｌ）を滴下して加えた。得られた混合物を室温に加温し、室温で１６時間撹拌した。続いて、飽和塩化アンモニウム水溶液を加え、混合物をＡｃＯＥｔで抽出した。次に合わせた有機抽出物を水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。分離された残渣をシリカゲルクロマトグラフィー（８０ｇ、ヘプタン／ＡｃＯＥｔ＝８０／２０～５０／５０）により精製して、表題化合物（９７ｍｇ、７％収率）を無色の油として得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ １．４２（ｓ，９Ｈ）２．４９－２．５８（ｍ，２Ｈ）３．４１（ｓ，３Ｈ）３．６９－３．７９（ｍ，２Ｈ）４．０５－４．１３（ｍ，２Ｈ）． 1.4 Synthesis of ADL1-H8, ADL1-H9, and ADL1-H10 1.4.1 Overview-General Procedure 4

Step 1: tert-butyl 3-hydroxypropanoate

To a mixture of sodium hydride (269 mg, 6.157 mmol) in DMF (30 mL) was added tert-butyl 3-hydroxypropanoate (0.909 mL, 6.157 mmol) at 0 ° C. The mixture was stirred at 0 ° C. for 1 hour, then methyl bromoacetate (0.624 mL, 6.157 mmol) was added dropwise. The resulting mixture was warmed to room temperature and stirred at room temperature for 16 hours. Subsequently, a saturated aqueous solution of ammonium chloride was added, and the mixture was extracted with AcOEt. The combined organic extracts were then washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The separated residue was purified by silica gel chromatography (80 g, heptane / AcOEt = 80/20 to 50/50) to give the title compound (97 mg, 7% yield) as a colorless oil.
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 1.42 (s, 9H) 2.49-2.58 (m, 2H) 3.41 (s, 3H) 3.69-3.79 (m, 3H) 2H) 4.05-4.13 (m, 2H).

ジクロロメタン（２ｍＬ）及びＴＦＡ（２ｍＬ）中の３－（２－メトキシ－２－オキソエトキシ）プロパン酸ｔｅｒｔ－ブチル（６６ｍｇ、０．３０２ｍｍｏｌ）の混合物を室温で３時間撹拌した。次にこの混合物を濃縮乾固して、表題化合物を無色の油として得た（５８ｍｇ、１００％）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ２．６２－２．７８（ｍ，２Ｈ）３．７６（ｓ，３Ｈ）３．７８－３．８５（ｍ，２Ｈ）４．１４（ｄ，Ｊ＝１．９５Ｈｚ，２Ｈ）． Step 2: 3- (2-Methoxy-2-oxoethoxy) propionic acid

A mixture of tert-butyl 3- (2-methoxy-2-oxoethoxy) propanoate (66 mg, 0.302 mmol) in dichloromethane (2 mL) and TFA (2 mL) was stirred at room temperature for 3 hours. The mixture was then concentrated to dryness to give the title compound as a colorless oil (58 mg, 100%).
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 2.62-2.78 (m, 2H) 3.76 (s, 3H) 3.78-3.85 (m, 2H) 4.14 (d, J = 1.95Hz, 2H).

ＴＨＦ（５ｍＬ）中の（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－３－メトキシ－４－（（トリエチルシリル）オキシ）ペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メタンアミン（１３３ｍｇ、０．２５４ｍｍｏｌ）と３－（２－メトキシ－２－オキソエトキシ）プロパン酸（６１．７ｍｇ、０．３８１ｍｍｏｌ）との混合物に室温でＨＡＴＵ（１４５ｍｇ、０．３８１ｍｍｏｌ）及びＤＩＰＥＡ（２２１μＬ、１．２７ｍｍｏｌ）を加えた。この混合物を１６時間撹拌し、次にＡｃＯＥｔで希釈した。有機層を分離し、水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。 Step 3: N-(((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-) 3-Methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl) -3- (2-Hydroxyethoxy) propenamide

((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -3-methoxy-) in THF (5 mL) 4-((Triethylsilyl) oxy) pentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran- HATU (145 mg, 0.381 mmol) in a mixture of 2-yl) methaneamine (133 mg, 0.254 mmol) and 3- (2-methoxy-2-oxoethoxy) propanoic acid (61.7 mg, 0.381 mmol) at room temperature. And DIPEA (221 μL, 1.27 mmol) were added. The mixture was stirred for 16 hours and then diluted with AcOEt. The organic layer was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo.

The separated residue was dissolved in THF (5 mL), cooled to 0 ° C., and then lithium borohydride (22.12 mg, 1.016 mmol) was added. The resulting mixture was stirred at the same temperature for 30 minutes, then warmed to room temperature and further stirred for 2 hours. Subsequently, a saturated aqueous solution of ammonium chloride and water were added, and the aqueous layer was extracted with AcOEt. The combined organic layers were washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The separated residue was then dissolved in THF (5 mL) and TBAF (1 M in THF solution, 0.508 mL, 0.508 mmol) was added at 0 ° C. After stirring at 0 ° C. for 30 minutes, the mixture was heated to room temperature and stirred for 2 hours. The mixture was subsequently diluted with AcOEt, the organic layer was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The obtained residue was purified by silica gel chromatography (24 g, heptane / AcOEt = 70/30 to 0/100, then AcOEt / MeOH = 80/20) to make the title compound (68 mg, 51% yield) colorless. Obtained as oil.
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.34 Hz, 3H) 0.85 (d, J = 6.83 Hz, 3H) 1.04 (d, J = 6. 34Hz, 3H) 1.16 (d, J = 6.8Hz, 4H) 1.19-1.22 (m, 2H) 1.27 (s, 3H) 1.42-1.65 (m, 4H) 1.71 (s, 3H) 1.79-1.92 (m, 2H) 2.37-2.50 (m, 4H) 2.54 (d, J = 9.27Hz, 2H) 2.89- 3.05 (m, 2H) 3.28 (d, J = 10.24Hz, 1H) 3.41 (td, J = 8.54, 2.44Hz, 1H) 3.52 (s, 3H) 3. 54-3.59 (m, 2H) 3.61-3.68 (m, 2H) 3.69-3.76 (m, 2H) 3.78-3.91 (m, 2H) 5.43- 5.52 (m, 1H) 5.92 (d, J = 10.73Hz, 1H) 6.24 (dd, J = 15.12, 10.73Hz, 1H) 6.77 (m, 1H).

Step 4: Synthesis of Carbamate N-(((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-(((2R, 3R) -3-(((2R, 3R) -3-(((S, 2E, 4E) -7-((2R, 3R) -3-(((S, 2E, 4E) -7- 2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro -2H-Pyran-2-yl) Methyl) -3- (2-hydroxyethoxy) propanamide (1.0 eq, 0.043 mmol) and 4-nitrophenyl chlorogiate (2.0 eq, 0.086 mmol) DMAP (5.0 eq) was added to the mixture with DIPEA (5.0 eq) at room temperature. The mixture was stirred at room temperature for 16 hours, then piperazine (10.0 eq) was added and the mixture was stirred for an additional hour. The resulting mixture was then diluted with dichloromethane, the organic layer was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by amino-functionalized silica gel chromatography (heptane / AcOEt = 50/50 to 0/100) to provide the desired compound.

２－（３－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－３－オキソプロポキシ）エチルピペラジン－１－カルボキシレート（Ｈ８）
セクション１．４．１に概説されるステップ１～４を用いて表題化合物を淡黄色の油として得た（１９．２ｍｇ、７０％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６３８．７８［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９６（ｄｄ，Ｊ＝１８．５４，６．３４Ｈｚ，１Ｈ）１．０３（ｄ，Ｊ＝５．８５Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝５．８５Ｈｚ，４Ｈ）１．１８－１．２５（ｍ，２Ｈ）１．２７（ｓ，３Ｈ）１．２９－１．３９（ｍ，１Ｈ）１．４１－１．６３（ｍ，４Ｈ）１．７０（ｓ，３Ｈ）１．７７－１．９７（ｍ，７Ｈ）２．４４（ｔ，Ｊ＝５．６１Ｈｚ，２Ｈ）２．４７－２．５７（ｍ，１Ｈ）２．８０（ｂｒ．ｓ．，４Ｈ）２．８５－２．９９（ｍ，１Ｈ）２．９９－３．０９（ｍ，１Ｈ）３．２７（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．３６－３．４８（ｍ，５Ｈ）３．５２（ｓ，３Ｈ）３．５４－３．５９（ｍ，１Ｈ）３．５９－３．６８（ｍ，２Ｈ）３．６８－３．７４（ｍ，２Ｈ）３．７７－３．９０（ｍ，１Ｈ）４．１５－４．２３（ｍ，２Ｈ）４．３６（ｔ，Ｊ＝６．１０Ｈｚ，１Ｈ）５．４４（ｄｄ，Ｊ＝１４．８８，８．５４Ｈｚ，１Ｈ）５．８８（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２３（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．４１（ｂｒ．ｓ．，１Ｈ）． 1.4.2 Synthesis of ADL1-H8 1.4.2.1 Synthesis of H8

2-(3-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy) -3-Methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl ) Amino) -3-oxopropoxy) Ethylpiperazin-1-carboxylate (H8)
The title compound was obtained as a pale yellow oil using steps 1-4 outlined in Section 1.4.1 (19.2 mg, 70% yield). LC / MS (ESI, m / z), 638.78 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.83 Hz, 3H) 0.85 (d, J = 6.83 Hz, 3H) 0.96 (dd, J = 18. 54,6.34Hz, 1H) 1.03 (d, J = 5.85Hz, 3H) 1.16 (d, J = 5.85Hz, 4H) 1.18-1.25 (m, 2H) 1. 27 (s, 3H) 1.29-1.39 (m, 1H) 1.41-1.63 (m, 4H) 1.70 (s, 3H) 1.77-1.97 (m, 7H) 2.44 (t, J = 5.61Hz, 2H) 2.47-2.57 (m, 1H) 2.80 (br.s., 4H) 2.85-2.99 (m, 1H) 2 .99-3.09 (m, 1H) 3.27 (d, J = 9.76Hz, 1H) 3.36-3.48 (m, 5H) 3.52 (s, 3H) 3.54-3 .59 (m, 1H) 3.59-3.68 (m, 2H) 3.68-3.74 (m, 2H) 3.77-3.90 (m, 1H) 4.15-4.23 (M, 2H) 4.36 (t, J = 6.10Hz, 1H) 5.44 (dd, J = 14.88, 8.54Hz, 1H) 5.88 (d, J = 10.73Hz, 1H) ) 6.23 (dd, J = 14.88, 10.98Hz, 1H) 6.41 (br.s., 1H).

１－（４－（（Ｓ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）４－（２－（３－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－３－オキソプロポキシ）エチル）ピペラジン－１，４－ジカルボキシレート（ＡＤＬ１－Ｈ８）
セクション１．２．７に概説されるものと同様の手順を用いて表題化合物を無色の油として得た（１８．３ｍｇ、６０％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３０Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９４（ｄｄ，Ｊ＝６．５９，４．１５Ｈｚ，７Ｈ）１．０１（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１０－１．２３（ｍ，４Ｈ）１．２５（ｓ，３Ｈ）１．２６－１．３２（ｍ，３Ｈ）１．４４－１．６５（ｍ，１０Ｈ）１．６８（ｓ，３Ｈ）１．７０－１．７５（ｍ，１Ｈ）１．８０－１．９２（ｍ，３Ｈ）２．００－２．０９（ｍ，１Ｈ）２．２５（ｔ，Ｊ＝７．５６Ｈｚ，２Ｈ）２．４１（ｔ，Ｊ＝５．８５Ｈｚ，２Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．９５（ｄｄ，Ｊ＝６．３４，４．３９Ｈｚ，１Ｈ）３．０８（ｄｔ，Ｊ＝１３．０５，６．４０Ｈｚ，１Ｈ）３．１４－３．２１（ｍ，１Ｈ）３．３８－３．４８（ｍ，１４Ｈ）３．４９（ｓ，４Ｈ）３．５８－３．６５（ｍ，２Ｈ）３．６９（ｔ，Ｊ＝６．１０Ｈｚ，２Ｈ）３．７２－３．８０（ｍ，１Ｈ）４．１３－４．１９（ｍ，３Ｈ）４．４８（ｄｄ，Ｊ＝９．０２，５．１２Ｈｚ，１Ｈ）５．０７（ｓ，２Ｈ）５．４６（ｄｄ，Ｊ＝１５．１２，９．２７Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．７６（ｓ，２Ｈ）７．３０（ｄ，Ｊ＝８．７８Ｈｚ，２Ｈ）７．５７（ｄ，Ｊ＝７．２５Ｈｚ，２Ｈ）． 1.4.2.2 Synthesis of H8

1-(4-((S) -2-((S) -2-(6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methyl) Butanamide) -5-Ureidopentanamide) benzyl) 4-(2-(3-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R)) -3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-Methyltetrahydro-2H-pyran-2-yl) methyl) amino) -3-oxopropoxy) ethyl) piperazin-1,4-dicarboxylate (ADL1-H8)
The title compound was obtained as a colorless oil using a procedure similar to that outlined in Section 1.2.7 (18.3 mg, 60% yield).
¹ H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.65 (d, J = 6.30 Hz, 3H) 0.80 (d, J = 6.83 Hz, 3H) 0.94 (dd, J = 6) .59, 4.15Hz, 7H) 1.01 (d, J = 6.83Hz, 3H) 1.08 (d, J = 6.34Hz, 3H) 1.10-1.23 (m, 4H) 1 .25 (s, 3H) 1.26-1.32 (m, 3H) 1.44-1.65 (m, 10H) 1.68 (s, 3H) 1.70-1.75 (m, 1H) ) 1.80-1.92 (m, 3H) 2.00-2.09 (m, 1H) 2.25 (t, J = 7.56Hz, 2H) 2.41 (t, J = 5.85Hz) , 2H) 2.63 (d, J = 9.27Hz, 1H) 2.95 (dd, J = 6.34, 4.39Hz, 1H) 3.08 (dt, J = 13.05, 6.40Hz , 1H) 3.14-3.21 (m, 1H) 3.38-3.48 (m, 14H) 3.49 (s, 4H) 3.58-3.65 (m, 2H) 3.69 (T, J = 6.10Hz, 2H) 3.72-3.80 (m, 1H) 4.13-4.19 (m, 3H) 4.48 (dd, J = 9.02, 5.12Hz) , 1H) 5.07 (s, 2H) 5.46 (dd, J = 15.12, 9.27Hz, 1H) 5.90 (d, J = 10.24Hz, 1H) 6.28 (dd, J) = 14.88, 10.98Hz, 1H) 6.76 (s, 2H) 7.30 (d, J = 8.78Hz, 2H) 7.57 (d, J = 7.25Hz, 2H).

２－（３－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－３－オキソプロポキシ）エチル１，４－ジアゼパン－１－カルボキシレート（Ｈ９）
セクション１．４．１に概説されるステップ１～４を用いて表題化合物を無色の油として得た（２１．１ｍｇ、７５％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６５２．８６［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）０．９２－１．００（ｍ，１Ｈ）１．０３（ｄ，Ｊ＝６．８Ｈ，３Ｈ）１．１６（ｄ，Ｊ＝６．８Ｈ，３Ｈ）１．２０－１．２４（ｍ，１Ｈ）１．２７（ｓ，４Ｈ）１．４７－１．６０（ｍ，４Ｈ）１．７０（ｓ，３Ｈ）１．７３－１．９０（ｍ，８Ｈ）２．３７－２．４７（ｍ，３Ｈ）２．５３（ｄ，Ｊ＝９．８Ｈｚ，１Ｈ）２．８１－２．８６（ｍ，２Ｈ）２．８７－２．９２（ｍ，２Ｈ）２．９４－２．９７（ｍ，１Ｈ）２．９９－３．０７（ｍ，１Ｈ）３．２７（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．３８－３．５０（ｍ，４Ｈ）３．５２（ｓ，３Ｈ）３．５５－３．５９（ｍ，１Ｈ）３．６１－３．６８（ｍ，２Ｈ）３．６８－３．７４（ｍ，２Ｈ）３．７７－３．９１（ｍ，１Ｈ）４．１９（ｂｒ．ｓ．，２Ｈ）４．３７（ｔ，Ｊ＝６．１０Ｈｚ，１Ｈ）５．４１－５．５０（ｍ，１Ｈ）５．８９（ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．２３（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．４６（ｄｄ，Ｊ＝３．１７，１．２２Ｈｚ，１Ｈ）． 1.4.3 Synthesis of ADL1-H9 1.4.3.1 Synthesis of H9

2-(3-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy) -3-Methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl ) Amino) -3-oxopropoxy) Ethyl 1,4-diazepan-1-carboxylate (H9)
The title compound was obtained as a colorless oil using steps 1-4 outlined in Section 1.4.1 (21.1 mg, 75% yield). LC / MS (ESI, m / z), 652.86 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.83 Hz, 3H) 0.85 (d, J = 7.32 Hz, 3H) 0.92-1.00 (m, 1H) 1.03 (d, J = 6.8H, 3H) 1.16 (d, J = 6.8H, 3H) 1.20-1.24 (m, 1H) 1.27 (s, 4H) 1.47-1.60 (m, 4H) 1.70 (s, 3H) 1.73-1.90 (m, 8H) 2.37-2.47 (m, 3H) 2.53 (d, 3H) J = 9.8Hz, 1H) 2.81-2.86 (m, 2H) 2.87-2.92 (m, 2H) 2.94-2.97 (m, 1H) 2.99-3. 07 (m, 1H) 3.27 (d, J = 9.76Hz, 1H) 3.38-3.50 (m, 4H) 3.52 (s, 3H) 3.55-3.59 (m, 1H) 3.61-3.68 (m, 2H) 3.68-3.74 (m, 2H) 3.77-3.91 (m, 1H) 4.19 (br.s., 2H) 4 .37 (t, J = 6.10Hz, 1H) 5.41-5.50 (m, 1H) 5.89 (d, J = 11.22Hz, 1H) 6.23 (dd, J = 14.88) , 10.98Hz, 1H) 6.46 (dd, J = 3.17, 1.22Hz, 1H).

１－（４－（（Ｓ）－２－（（Ｓ）－２－（６－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）ヘキサンアミド）－３－メチルブタンアミド）－５－ウレイドペンタンアミド）ベンジル）４－（２－（３－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－３－オキソプロポキシ）エチル）１，４－ジアゼパン－１，４－ジカルボキシレート（ＡＤＬ１－Ｈ９）
セクション１．２．７に概説されるものと同様の手順を用いて表題化合物を得た（２５．２ｍｇ、７２％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３０Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９３（ｔ，Ｊ＝５．８５Ｈｚ，７Ｈ）１．０１（ｄ，Ｊ＝６．３０Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３０Ｈｚ，３Ｈ）１．１１－１．２１（ｍ，２Ｈ）１．２１－１．３２（ｍ，７Ｈ）１．４３－１．６５（ｍ，１０Ｈ）１．６９（ｓ，３Ｈ）１．７０－１．９１（ｍ，５Ｈ）２．０５（ｑ，Ｊ＝６．８Ｈｚ，１Ｈ）２．２５（ｔ，Ｊ＝７．３２Ｈｚ，２Ｈ）２．３６－２．４８（ｍ，３Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．９５（ｄｄ，Ｊ＝５．８５，４．３９Ｈｚ，１Ｈ）３．０３－３．１３（ｍ，２Ｈ）３．１３－３．２１（ｍ，１Ｈ）３．３７－３．４８（ｍ，７Ｈ）３．４８－３．５９（ｍ，８Ｈ）３．６２－３．７０（ｍ，２Ｈ）３．７６（ｑｕｉｎ，Ｊ＝６．４６Ｈｚ，１Ｈ）４．１１－４．２２（ｍ，２Ｈ）４．４７－４．５３（ｍ，１Ｈ）５．０２－５．０９（ｍ，２Ｈ）５．４５（ｄｄ，Ｊ＝１４．８８，９．０３Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．６４，１１．２２Ｈｚ，１Ｈ）６．７６（ｓ，２Ｈ）７．２６－７．３７（ｍ，２Ｈ）７．５７（ｄ，Ｊ＝７．８１Ｈｚ，２Ｈ）． 1.4.3.2 Synthesis of ADL1-H9

1-(4-((S) -2-((S) -2-(6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexaneamide) -3-methyl) Butanamide) -5-Ureidopentanamide) benzyl) 4-(2-(3-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R)) -3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-Methyltetrahydro-2H-pyran-2-yl) methyl) amino) -3-oxopropoxy) ethyl) 1,4-diazepan-1,4-dicarboxylate (ADL1-H9)
The title compound was obtained using a procedure similar to that outlined in Section 1.2.7 (25.2 mg, 72% yield).
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.65 (d, J = 6.30 Hz, 3H) 0.80 (d, J = 6.83 Hz, 3H) 0.93 (t, J = 5) .85Hz, 7H) 1.01 (d, J = 6.30Hz, 3H) 1.08 (d, J = 6.30Hz, 3H) 1.11-1.21 (m, 2H) 1.21-1 .32 (m, 7H) 1.43-1.65 (m, 10H) 1.69 (s, 3H) 1.70-1.91 (m, 5H) 2.05 (q, J = 6.8Hz) , 1H) 2.25 (t, J = 7.32Hz, 2H) 2.36-2.48 (m, 3H) 2.63 (d, J = 9.27Hz, 1H) 2.95 (dd, J) = 5.85, 4.39Hz, 1H) 3.03-3.13 (m, 2H) 3.13-3.21 (m, 1H) 3.37-3.48 (m, 7H) 3.48 -3.59 (m, 8H) 3.62-3.70 (m, 2H) 3.76 (quin, J = 6.46Hz, 1H) 4.11-4.22 (m, 2H) 4.47 -4.53 (m, 1H) 5.02-5.09 (m, 2H) 5.45 (dd, J = 14.88, 9.03Hz, 1H) 5.90 (d, J = 10.73Hz) , 1H) 6.28 (dd, J = 14.64, 11.22Hz, 1H) 6.76 (s, 2H) 7.26-7.37 (m, 2H) 7.57 (d, J = 7) .81Hz, 2H).

２－（３－（（（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メチル）アミノ）－３－オキソプロポキシ）エチル３－（メチルアミノ）ピロリジン－１－カルボキシレート（Ｈ１０）
セクション１．４．１に概説されるステップ１～４を用いて表題化合物を無色の油として得た（２４．８ｍｇ、８８％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６５２．９１［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９６（ｄｄ，Ｊ＝１８．７８，６．５９Ｈｚ，１Ｈ）１．０３（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１８－１．２５（ｍ，３Ｈ）１．２７（ｓ ３Ｈ）１．２０－１．３２（ｍ，５Ｈ）１．４１－１．６３（ｍ，４Ｈ）１．７０（ｓ，３Ｈ）１．７２－１．９０（ｍ，４Ｈ）２．０２（ｄｄ，Ｊ＝１２．４４，６．１０Ｈｚ，２Ｈ）２．４１（ｓ ３Ｈ）２．４３－２．５０（ｍ，２Ｈ）２．５４（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）２．９５（ｔ，Ｊ＝５．３７Ｈｚ，１Ｈ）３．０１－３．１４（ｍ，１Ｈ）３．１８－３．２４（ｍ，１Ｈ）３．２７（ｄ，Ｊ＝９．７Ｈｚ，１Ｈ）３．３８－３．４９（ｍ，２Ｈ）３．５２（ｓ，６Ｈ）３．６１－３．６５（ｍ，２Ｈ）３．６９－３．７４（ｍ，２Ｈ）３．８３（ｑｕｉｎ，Ｊ＝５．９８Ｈｚ，１Ｈ）４．１５－４．２２（ｍ，１Ｈ）４．３４（ｔ，Ｊ＝６．１０Ｈｚ，１Ｈ）５．４４（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８８（ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．２３（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．４３－６．５１（ｍ，１Ｈ）． 1.4.4 Synthesis of ADL1-H10 1.4.4.1 Synthesis of H10

2-(3-((((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy) -3-Methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) methyl ) Amino) -3-oxopropoxy) Ethyl 3- (methylamino) pyrrolidine-1-carboxylate (H10)
The title compound was obtained as a colorless oil using steps 1-4 outlined in Section 1.4.1 (24.8 mg, 88% yield). LC / MS (ESI, m / z), 652.91 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.34 Hz, 3H) 0.85 (d, J = 6.83 Hz, 3H) 0.96 (dd, J = 18. 78, 6.59Hz, 1H) 1.03 (d, J = 6.8Hz, 3H) 1.16 (d, J = 6.34Hz, 3H) 1.18-1.25 (m, 3H) 1. 27 (s 3H) 1.20-1.32 (m, 5H) 1.41-1.63 (m, 4H) 1.70 (s, 3H) 1.72-1.90 (m, 4H) 2 .02 (dd, J = 12.44, 6.10Hz, 2H) 2.41 (s 3H) 2.43-2.50 (m, 2H) 2.54 (d, J = 9.76Hz, 1H) 2.95 (t, J = 5.37Hz, 1H) 3.01-3.14 (m, 1H) 3.18-3.24 (m, 1H) 3.27 (d, J = 9.7Hz, 1H) 3.38-3.49 (m, 2H) 3.52 (s, 6H) 3.61-3.65 (m, 2H) 3.69-3.74 (m, 2H) 3.83 ( quin, J = 5.98Hz, 1H) 4.15-4.22 (m, 1H) 4.34 (t, J = 6.10Hz, 1H) 5.44 (dd, J = 15.12, 8. 78Hz, 1H) 5.88 (d, J = 11.22Hz, 1H) 6.23 (dd, J = 14.88, 10.98Hz, 1H) 6.43-6.51 (m, 1H).

セクション１．２．７に概説されるものと同様の手順を用いて表題化合物を無色の油として得た（２２．１ｍｇ、５７％収率）。
１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ４）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９４（ｄｄ，Ｊ＝６．３４，４．８８Ｈｚ，８Ｈ）１．０１（ｄｄ，Ｊ＝３．９０，２．９３Ｈｚ，４Ｈ）１．０８（ｄ，Ｊ＝６．３０Ｈｚ，３Ｈ）１．１１－１．２２（ｍ，３Ｈ）１．２２－１．３１（ｍ，７Ｈ）１．４４－１．６５（ｍ，１０Ｈ）１．６８（ｓ ３Ｈ）１．７１－１．９１（ｍ，４Ｈ）１．９９－２．０９（ｍ，３Ｈ）２．２５（ｔ，Ｊ＝７．５６Ｈｚ，２Ｈ）２．４１（ｔ，Ｊ＝５．８５Ｈｚ，２Ｈ）２．６２（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．８４（ｍ，４Ｈ）２．９３－２．９９（ｍ，２Ｈ）３．０４－３．１３（ｍ，２Ｈ）３．１３－３．２１（ｍ，１Ｈ）３．４３（ｔ，Ｊ＝７．０７Ｈｚ，２Ｈ）３．５０（ｓ，３Ｈ）３．５２－３．５６（ｍ，２Ｈ）３．６１（ｔ，Ｊ＝４．３９Ｈｚ，２Ｈ）３．６９（ｔ，Ｊ＝５．３７Ｈｚ，２Ｈ）３．７３－３．７９（ｍ，１Ｈ）４．１５（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）４．４９（ｄｄ，Ｊ＝９．０３，５．１２Ｈｚ，１Ｈ）５．０６（ｓ，２Ｈ）５．４５（ｄｄ，Ｊ＝１４．８８，９．０３Ｈｚ，１Ｈ）５．９０（ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）６．７６（ｓ，Ｊ＝４．５６Ｈｚ，２Ｈ）７．３０（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）７．５７（ｄ，Ｊ＝８．２９Ｈｚ，２Ｈ）． 1.4.4.2 Synthesis of ADL1-H10

The title compound was obtained as a colorless oil using a procedure similar to that outlined in Section 1.2.7 (22.1 mg, 57% yield).
1H NMR (400MHz, methanol-d4) δ ppm 0.65 (d, J = 6.34Hz, 3H) 0.80 (d, J = 6.83Hz, 3H) 0.94 (dd, J = 6.34) , 4.88Hz, 8H) 1.01 (dd, J = 3.90, 2.93Hz, 4H) 1.08 (d, J = 6.30Hz, 3H) 1.11-1.22 (m, 3H) ) 1.22-1.31 (m, 7H) 1.44-1.65 (m, 10H) 1.68 (s 3H) 1.71-1.91 (m, 4H) 1.99-2. 09 (m, 3H) 2.25 (t, J = 7.56Hz, 2H) 2.41 (t, J = 5.85Hz, 2H) 2.62 (d, J = 9.27Hz, 1H) 2. 84 (m, 4H) 2.93-2.99 (m, 2H) 3.04-3.13 (m, 2H) 3.13-3.21 (m, 1H) 3.43 (t, J = 7.07Hz, 2H) 3.50 (s, 3H) 3.52-3.56 (m, 2H) 3.61 (t, J = 4.39Hz, 2H) 3.69 (t, J = 5. 37Hz, 2H) 3.73-3.79 (m, 1H) 4.15 (d, J = 7.32Hz, 3H) 4.49 (dd, J = 9.03, 5.12Hz, 1H) 5. 06 (s, 2H) 5.45 (dd, J = 14.88, 9.03Hz, 1H) 5.90 (d, J = 11.22Hz, 1H) 6.28 (dd, J = 14.88, 10.98Hz, 1H) 6.76 (s, J = 4.56Hz, 2H) 7.30 (d, J = 8.29Hz, 2H) 7.57 (d, J = 8.29Hz, 2H).

２－（（２Ｒ，４Ｒ，５Ｓ，６Ｓ）－４－（（４－（３－（２－（２－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）エトキシ）エトキシ）プロパノイル）ピペラジン－１－カルボニル）オキシ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）酢酸（ＡＤＬ２－Ｈ１）
ＤＭＦ（１ｍＬ）中のＨ１（例えば、セクション１．２．２を参照のこと；２２．５ｍｇ、０．０３ｍｍｏｌ）の混合物に２，５－ジオキソピロリジン－１－イル３－｛２－［２－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）エトキシ］エトキシ｝プロパノエート（１０．５５ｍｇ、０．０３ｍｍｏｌ）及びＤＩＰＥＡ（０．０１６ｍＬ、０．０８９ｍｍｏｌ）を加えた。得られた混合物を室温で４時間撹拌した。次に、溶媒を真空除去し、得られた残渣を逆相クロマトグラフィー（ＯＤＳ、２４ｇ、Ｈ_２Ｏ／ＭｅＣＮ＝９５／５～６０／４０）により精製して、表題化合物を無色の油として得た（１５．７ｍｇ、６５％収率）。
^１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ_４）δ ｐｐｍ ０．７３（ｄ，Ｊ＝６．３４Ｈｚ，４Ｈ）０．９７－１．１５（ｍ，１３Ｈ）１．２１（ｓ，２Ｈ）１．２８－１．４１（ｍ，１Ｈ）１．４２－１．６２（ｍ，２Ｈ）１．６９（ｄ，Ｊ＝４．３９Ｈｚ，４Ｈ）２．０８－２．２０（ｍ，１Ｈ）２．３３－２．３９（ｍ，１Ｈ）２．４２－２．５８（ｍ，３Ｈ）２．６３（ｔ，Ｊ＝５．３０Ｈｚ，２Ｈ）２．９１－２．９４（ｍ，１Ｈ）３．３３（ｄ，Ｊ＝３．４１Ｈｚ，１Ｈ）３．３６（ｄ，Ｊ＝１．９５Ｈｚ，２Ｈ）３．４４－３．６０（ｍ，１８Ｈ）３．６０－３．６７（ｍ，４Ｈ）３．６９（ｔ，Ｊ＝５．３７Ｈｚ，２Ｈ）３．７５－３．７８（ｍ，１Ｈ）３．８１－４．００（ｍ，２Ｈ）４．２２－４．３１（ｍ，１Ｈ）４．５２－４．６０（ｍ，１Ｈ）５．５８－５．７２（ｍ，１Ｈ）５．８９－６．００（ｍ，１Ｈ）６．１３－６．３１（ｍ，１Ｈ）６．７１－６．８９（ｍ，２Ｈ）． 1.5 Synthesis of ADL2-H1

2-((2R, 4R, 5S, 6S) -4-((4- (3- (2- (2- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)) ethoxy ) Ethoxy) propanoyl) piperazine-1-carbonyl) oxy) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3) -Methoxypentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl) acetic acid (ADL2) -H1)
2,5-Dioxopyrrolidine-1-yl 3- {2- [2] in a mixture of H1 (see, eg, Section 1.2.2; 22.5 mg, 0.03 mmol) in DMF (1 mL). -(2,5-Dioxo-2,5-dihydro-1H-pyrrole-1-yl) ethoxy] ethoxy} propanoate (10.55 mg, 0.03 mmol) and DIPEA (0.016 mL, 0.089 mmol) were added. .. The resulting mixture was stirred at room temperature for 4 hours. The solvent was then evacuated and the resulting residue was purified by reverse phase chromatography (ODS, 24 g, H ₂ O / MeCN = 95/5-60/40) to give the title compound as a colorless oil. (15.7 mg, 65% yield).
^{1 1} H NMR (400 MHz, methanol-d ₄ ) δ ppm 0.73 (d, J = 6.34 Hz, 4H) 0.97-1.15 (m, 13H) 1.21 (s, 2H) 1.28 -1.41 (m, 1H) 1.42-1.62 (m, 2H) 1.69 (d, J = 4.39Hz, 4H) 2.08-2.20 (m, 1H) 2.33 -2.39 (m, 1H) 2.42-2.58 (m, 3H) 2.63 (t, J = 5.30Hz, 2H) 2.91-2.94 (m, 1H) 3.33 (D, J = 3.41Hz, 1H) 3.36 (d, J = 1.95Hz, 2H) 3.44-3.60 (m, 18H) 3.60-3.67 (m, 4H) 3 .69 (t, J = 5.37Hz, 2H) 3.75-3.78 (m, 1H) 3.81-4.00 (m, 2H) 4.22-4.31 (m, 1H) 4 .52-4.60 (m, 1H) 5.58-5.72 (m, 1H) 5.89-6.00 (m, 1H) 6.13-6.31 (m, 1H) 6.71 -6.89 (m, 2H).

ＴＨＦ（１ｍＬ）中の（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－３－メトキシ－４－（（トリエチルシリル）オキシ）ペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）メタンアミン（３２ｍｇ、０．０６１ｍｍｏｌ）の混合物にＴＢＡＦ（ＴＨＦ中１Ｍ溶液、０．１８３ｍＬ、０．１８３ｍｍｏｌ）を加えた。得られた混合物を室温で２時間撹拌し、次にＡｃＯＥｔで希釈した。有機相を分離し、水及びブラインで洗浄し、硫酸ナトリウムで乾燥させて、ろ過し、真空濃縮した。分離された残渣をアミノ官能化シリカゲルクロマトグラフィー（２４ｇ、ヘプタン／ＡｃＯＥｔ＝５０／５０～０／１００、次にＡｃＯＥｔ／ＭｅＯＨ＝８０／２０）により精製すると、表題化合物が無色の油として提供された（１５．１ｍｇ、６０．４％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），４１０．０７［Ｍ＋Ｈ］^＋．
^１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｄ，Ｊ＝６．８０Ｈｚ，３Ｈ）０．８６（ｄ，Ｊ＝６．８０Ｈｚ，３Ｈ）１．０３（ｄ，Ｊ＝６．８０Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．３０Ｈｚ，４Ｈ）１．１９－１．２４（ｍ，２Ｈ）１．２７（ｓ，３Ｈ）１．２９－１．３８（ｍ，１Ｈ）１．４４－１．５７（ｍ，３Ｈ）１．６２－１．６６（ｍ，１Ｈ）１．７１（ｓ，５Ｈ）１．７４－１．９３（ｍ，３Ｈ）２．３４－２．４５（ｍ，１Ｈ）２．５４（ｄ，Ｊ＝９．００Ｈｚ，１Ｈ）２．６２－２．７４（ｍ，２Ｈ）２．９２－２．９８（ｍ，１Ｈ）３．２４－３．３２（ｍ，２Ｈ）３．５２（ｓ，３Ｈ）３．７９－３．８８（ｍ，１Ｈ）５．３９－５．４８（ｍ，１Ｈ）５．８８（ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２３（ｄｄｄ，Ｊ＝１４．６４，１１．２２，３．４１Ｈｚ，１Ｈ）． 1.6 Synthesis of ADL2-H11 Step 1: (2R, 3R, 4R) -4-((2R, 3R) -3-((S, 3E, 5E) -6-((2S, 3S, 6R)-)- 6- (Aminomethyl) -3-methyltetrahydro-2-H-pyran-2-yl) -2-methylhepta-3,5-diene-1-yl) -3-methyloxylan-2-yl) -3-methoxypentane -Synthesis of 2-all (H11)

((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -3-methoxy-) in THF (1 mL) 4-((Triethylsilyl) oxy) pentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2-H-pyran- TBAF (1M solution in THF, 0.183 mL, 0.183 mmol) was added to the mixture of 2-yl) methaneamine (32 mg, 0.061 mmol). The resulting mixture was stirred at room temperature for 2 hours and then diluted with AcOEt. The organic phase was separated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The separated residue was purified by amino-functionalized silica gel chromatography (24 g, heptane / AcOEt = 50/50 to 0/100, then AcOEt / MeOH = 80/20) to provide the title compound as a colorless oil. (15.1 mg, 60.4% yield). LC / MS (ESI, m / z), 410.07 [M + H] ⁺ .
^{1 1} H NMR (400 MHz, chloroform-d) δ ppm 0.65 (d, J = 6.80 Hz, 3H) 0.86 (d, J = 6.80 Hz, 3H) 1.03 (d, J = 6. 80Hz, 3H) 1.16 (d, J = 6.30Hz, 4H) 1.19-1.24 (m, 2H) 1.27 (s, 3H) 1.29-1.38 (m, 1H) 1.44-1.57 (m, 3H) 1.62-1.66 (m, 1H) 1.71 (s, 5H) 1.74-1.93 (m, 3H) 2.34-2. 45 (m, 1H) 2.54 (d, J = 9.00Hz, 1H) 2.62-2.74 (m, 2H) 2.92-2.98 (m, 1H) 3.24-3. 32 (m, 2H) 3.52 (s, 3H) 3.79-3.88 (m, 1H) 5.39-5.48 (m, 1H) 5.88 (d, J = 10.73Hz, 1H) 6.23 (ddd, J = 14.64, 11.22, 3.41Hz, 1H).

ＤＭＦ（１ｍＬ、１２．９１５ｍｍｏｌ）中のステップ１から得られた化合物（１５．１ｍｇ、０．０３７ｍｍｏｌ）の混合物に２，５－ジオキソピロリジン－１－イル３－｛２－［２－（２，５－ジオキソ－２，５－ジヒドロ－１Ｈ－ピロール－１－イル）エトキシ］エトキシ｝プロパノエート（１３．１ｍｇ、０．０３７ｍｍｏｌ）及びＤＩＰＥＡ（０．０１９ｍｌ、０．１１１ｍｍｏｌ）を加えた。得られた混合物を室温で１６時間撹拌した。続いて、この混合物を真空濃縮し、得られた残渣を逆相クロマトグラフィー（ＯＤＳ、２４ｇ、Ｈ_２Ｏ／ＭｅＣＮ＝９５／５～６０／４０）により精製して、表題化合物を無色の油として得た（１４．９ｍｇ、６２％収率）。 Step 2: 3- (2- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) propoxy) ethoxy) -N-(((2R, 5S, 6S) -6) -((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2) -Il) -6-Methylhepta-2,4-dien-2-yl) -5-Methyltetrahydro-2H-pyran-2-yl) Methyl) Prosanamide (ADL2-H11) synthesis

A mixture of compounds (15.1 mg, 0.037 mmol) obtained from step 1 in DMF (1 mL, 12.915 mmol) with 2,5-dioxopyrrolidine-1-yl 3- {2- [2- (2). , 5-Dioxo-2,5-dihydro-1H-pyrrole-1-yl) ethoxy] ethoxy} propanoate (13.1 mg, 0.037 mmol) and DIPEA (0.019 ml, 0.111 mmol) were added. The resulting mixture was stirred at room temperature for 16 hours. Subsequently, the mixture was concentrated in vacuo and the resulting residue was purified by reverse phase chromatography (ODS, 24 g, H2 O / MeCN ₌ 95/5-60/40) to give the title compound as a colorless oil. Obtained (14.9 mg, 62% yield).

ＤＭＦ（０．０２８Ｍ）中の２，５－ジオキソピロリジン－１－イル２－（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－４－ヒドロキシ－３－メトキシペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－イル）アセテート（１．０当量）の溶液にアミン（１６ｍｇ、０．０５６ｍｍｏｌ）及びＤＩＰＥＡ（２．０当量）を加えた。この反応混合物を室温で１６時間撹拌し、次に混合物をＨＰＬＣにより精製すると、所望の生成物が提供された。 1.7 Synthesis of H18, H20, H23, H16, H15, H24, H13, H14, H17, H19, and H21 H18, H20, H23, H16, H15, H24, H13, H14, H17, H19, and H21 Prepared by the following general procedure (general procedure 1.7).

2,5-Dioxopyrrolidine-1-yl2-((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R)-) in DMF (0.028M) 3-((2R, 3R, 4R) -4-hydroxy-3-methoxypentane-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl)- Amine (16 mg, 0.056 mmol) and DIPEA (2.0 eq) were added to a solution of 5-methyltetrahydro-2H-pyran-2-yl) acetate (1.0 eq). The reaction mixture was stirred at room temperature for 16 hours and then purified by HPLC to provide the desired product.

一般的手順１．７を用いて、Ｈ２０を無色のアモルファス生成物として得た（１５．７ｍｇ、０．０２２ｍｍｏｌ、７９％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），７０７．９９［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８５（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０４（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１６（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１８－１．２５（ｍ，２Ｈ）１．２７（ｓ，４Ｈ）１．３０－１．５９（ｍ，８Ｈ）１．７１－１．８０（ｍ，２Ｈ）１．８１－１．８９（ｍ，２Ｈ）２．３５（ｂｒ ｄ，Ｊ＝３．９０Ｈｚ，２Ｈ）２．３８－２．４４（ｍ，１Ｈ）２．５２－２．６０（ｍ，４Ｈ）２．７９－２．８６（ｍ，４Ｈ）２．９６（ｔ，Ｊ＝５．３７Ｈｚ，１Ｈ）３．０６－３．１６（ｍ，１Ｈ）３．２４－３．３０（ｍ，１Ｈ）３．３２（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．５２（ｓ，３Ｈ）３．６４（ｂｒ ｄ，Ｊ＝４．３９Ｈｚ，５Ｈ）３．７１（ｓ，３Ｈ）３．８３（ｔ，Ｊ＝５．８５Ｈｚ，１Ｈ）４．３０－４．４２（ｍ，１Ｈ）５．４０－５．５３（ｍ，３Ｈ）５．８９（ｂｒ ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．１６－６．２８（ｍ，１Ｈ）６．７５（ｂｒ ｔ，Ｊ＝５．８５Ｈｚ，１Ｈ）． 1.7.1 Synthesis of H20

General procedure 1.7 was used to give H20 as a colorless amorphous product (15.7 mg, 0.022 mmol, 79% yield). LC / MS (ESI, m / z), 707.99 [M + H] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.66 (d, J = 6.83Hz, 3H) 0.85 (d, J = 6.83Hz, 3H) 1.04 (d, J = 6.83Hz) , 3H) 1.16 (d, J = 6.34Hz, 3H) 1.18-1.25 (m, 2H) 1.27 (s, 4H) 1.30-1.59 (m, 8H) 1 .71-1.80 (m, 2H) 1.81-1.89 (m, 2H) 2.35 (br d, J = 3.90Hz, 2H) 2.38-2.44 (m, 1H) 2.52-2.60 (m, 4H) 2.79-2.86 (m, 4H) 2.96 (t, J = 5.37Hz, 1H) 3.06-3.16 (m, 1H) 3.24-3.30 (m, 1H) 3.32 (d, J = 9.76Hz, 1H) 3.52 (s, 3H) 3.64 (br d, J = 4.39Hz, 5H) 3 .71 (s, 3H) 3.83 (t, J = 5.85Hz, 1H) 4.30-4.42 (m, 1H) 5.40-5.53 (m, 3H) 5.89 (br) d, J = 10.73Hz, 1H) 6.16-6.28 (m, 1H) 6.75 (br t, J = 5.85Hz, 1H).

一般的手順１．７からＨ２３を得た（６．９ｍｇ、３７％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６６５．９６［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ４）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０２（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１２－１．２３（ｍ，２Ｈ）１．２５（ｓ，４Ｈ）１．３１－１．３７（ｍ，１Ｈ）１．４３－１．５５（ｍ，２Ｈ）１．６３（ｂｒ ｄ，Ｊ＝１３．１７Ｈｚ，１Ｈ）１．６７（ｓ，３Ｈ）１．８１－１．９３（ｍ，３Ｈ）２．２１－２．４０（ｍ，１Ｈ）２．６３（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）２．７４（ｓ，２Ｈ）２．９２－２．９７（ｍ，１Ｈ）３．０５（ｂｒ ｓ，３Ｈ）３．１２－３．２２（ｍ，１Ｈ）３．３１－３．３８（ｍ，２Ｈ）３．５０（ｓ，３Ｈ）３．６３（ｂｒ ｓ，４Ｈ）３．６８－３．７８（ｍ，２Ｈ）４．２３（ｂｒ ｄ，Ｊ＝４．３９Ｈｚ，１Ｈ）５．４５（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８９（ｂｒ ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２７（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）． 1.7.2 Synthesis of H23

H23 was obtained from the general procedure 1.7 (6.9 mg, 37% yield). LC / MS (ESI, m / z), 665.96 [M + H] ⁺ .
1H NMR (400MHz, methanol-d4) δ ppm 0.66 (d, J = 6.34Hz, 3H) 0.80 (d, J = 6.83Hz, 3H) 1.02 (d, J = 6.34Hz) , 3H) 1.08 (d, J = 6.34Hz, 3H) 1.12-1.23 (m, 2H) 1.25 (s, 4H) 1.31-1.37 (m, 1H) 1 .43-1.55 (m, 2H) 1.63 (br d, J = 13.17Hz, 1H) 1.67 (s, 3H) 1.81-1.93 (m, 3H) 2.21- 2.40 (m, 1H) 2.63 (d, J = 9.76Hz, 1H) 2.74 (s, 2H) 2.92-2.97 (m, 1H) 3.05 (br s, 3H) ) 3.12-3.22 (m, 1H) 3.31-3.38 (m, 2H) 3.50 (s, 3H) 3.63 (br s, 4H) 3.68-3.78 ( m, 2H) 4.23 (br d, J = 4.39Hz, 1H) 5.45 (dd, J = 15.12, 8.78Hz, 1H) 5.89 (br d, J = 10.73Hz, 1H) 6.27 (dd, J = 14.88, 10.98Hz, 1H).

一般的手順１．７を用いてＨ１６を無色のアモルファス生成物として得た（６．４ｍｇ、３４％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６７９．８６［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６７（ｂｒ ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｂｒ ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０１－１．０６（ｍ，３Ｈ）１．１４－１．１９（ｍ，３Ｈ）１．１９－１．２８（ｍ，５Ｈ）１．４３（ｂｒ ｄｄ，Ｊ＝１２．２０，９．７６Ｈｚ，１Ｈ）１．４７－１．５５（ｍ，１Ｈ）１．６１－１．６４（ｍ，１Ｈ）１．７１（ｂｒ ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）１．８３－１．９０（ｍ，２Ｈ）２．０３－２．２０（ｍ，１Ｈ）２．３７－２．４４（ｍ，３Ｈ）２．４９－２．５６（ｍ，５Ｈ）２．７５（ｂｒ ｓ，１Ｈ）２．７８－２．８４（ｍ，３Ｈ）２．８７－２．９４（ｍ，１Ｈ）２．９４－２．９８（ｍ，１Ｈ）３．３５（ｂｒ ｄｄ，Ｊ＝１３．４１，１０．００Ｈｚ，２Ｈ）３．５２（ｓ，４Ｈ）３．６０（ｂｒ ｓ，４Ｈ）３．６９（ｂｒ ｓ，３Ｈ）３．７１（ｓ，４Ｈ）３．８３（ｔｄ，Ｊ＝１２．５６，６．５９Ｈｚ，２Ｈ）４．０６－４．２０（ｍ，１Ｈ）４．５７－４．６８（ｍ，１Ｈ）５．４７（ｔｄ，Ｊ＝１５．９８，８．５４Ｈｚ，１Ｈ）５．８１－５．９６（ｍ，１Ｈ）６．１４－６．３６（ｍ，１Ｈ）． 1.7.3 Synthesis of H16

H16 was obtained as a colorless amorphous product using general procedure 1.7 (6.4 mg, 34% yield). LC / MS (ESI, m / z), 679.86 [M + H] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.67 (br d, J = 6.34Hz, 3H) 0.85 (br d, J = 6.83Hz, 3H) 1.01-1.06 (m) , 3H) 1.14-1.19 (m, 3H) 1.19-1.28 (m, 5H) 1.43 (br dd, J = 12.20, 9.76Hz, 1H) 1.47- 1.55 (m, 1H) 1.61-1.64 (m, 1H) 1.71 (br d, J = 7.32Hz, 3H) 1.83-1.90 (m, 2H) 2.03 -2.20 (m, 1H) 2.37-2.44 (m, 3H) 2.49-2.56 (m, 5H) 2.75 (br s, 1H) 2.78-2.84 ( m, 3H) 2.87-2.94 (m, 1H) 2.94-2.98 (m, 1H) 3.35 (br dd, J = 13.41, 10.00Hz, 2H) 3.52 (S, 4H) 3.60 (br s, 4H) 3.69 (br s, 3H) 3.71 (s, 4H) 3.83 (td, J = 12.56, 6.59Hz, 2H) 4 .06-4.20 (m, 1H) 4.57-4.68 (m, 1H) 5.47 (td, J = 15.98, 8.54Hz, 1H) 5.81-5.96 (m) , 1H) 6.14-6.36 (m, 1H).

一般的手順１．７により、Ｈ１６の合成中の副生成物としてＨ１５を得た（６．６ｍｇ、３５％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６６５．４６［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ４）δ ｐｐｍ ０．６６（ｂｒ ｄ，Ｊ＝６．３４Ｈｚ，４Ｈ）０．８１（ｂｒ ｄ，Ｊ＝５．８５Ｈｚ，３Ｈ）０．９９－１．０４（ｍ，４Ｈ）１．０７－１．１０（ｍ，４Ｈ）１．１７（ｂｒ ｓ，１Ｈ）１．１９－１．２７（ｍ，５Ｈ）１．４６－１．５５（ｍ，２Ｈ）１．６５（ｂｒ ｓ，１Ｈ）１．６８（ｓ，４Ｈ）１．８２－１．９５（ｍ，２Ｈ）２．００－２．１８（ｍ，１Ｈ）２．２９－２．４９（ｍ，４Ｈ）２．６１－２．６７（ｍ，４Ｈ）２．９２－３．０３（ｍ，５Ｈ）３．３４（ｂｒ ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）３．５０（ｓ，３Ｈ）３．５８－３．７７（ｍ，８Ｈ）４．２８（ｔ，Ｊ＝９．７６Ｈｚ，１Ｈ）５．４０－５．５３（ｍ，１Ｈ）５．８３－５．９９（ｍ，１Ｈ）６．２８（ｄｄ，Ｊ＝１４．６３，１０．７３Ｈｚ，１Ｈ）． 1.7.4 Synthesis of H15

General procedure 1.7 gave H15 as a by-product during the synthesis of H16 (6.6 mg, 35% yield). LC / MS (ESI, m / z), 665.46 [M + H] ⁺ .
1H NMR (400MHz, methanol-d4) δ ppm 0.66 (br d, J = 6.34Hz, 4H) 0.81 (br d, J = 5.85Hz, 3H) 0.99-1.04 (m) , 4H) 1.07-1.10 (m, 4H) 1.17 (br s, 1H) 1.19-1.27 (m, 5H) 1.46-1.55 (m, 2H) 1. 65 (br s, 1H) 1.68 (s, 4H) 1.82-1.95 (m, 2H) 2.00-2.18 (m, 1H) 2.29-2.49 (m, 4H) ) 2.61-2.67 (m, 4H) 2.92-3.03 (m, 5H) 3.34 (br d, J = 10.24Hz, 1H) 3.50 (s, 3H) 3. 58-3.77 (m, 8H) 4.28 (t, J = 9.76Hz, 1H) 5.40-5.53 (m, 1H) 5.83-5.99 (m, 1H) 6. 28 (dd, J = 14.63, 10.73Hz, 1H).

一般的手順１．７を用いてＨ２４を無色のアモルファス生成物として得た（１２．１ｍｇ、９４％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６９３．７２［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ４）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０２（ｄ，Ｊ＝６．８３Ｈｚ，４Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，４Ｈ）１．３２－１．３７（ｍ，８Ｈ）１．４４－１．５０（ｍ，４Ｈ）１．６５－１．６８（ｍ，５Ｈ）１．７５－１．９６（ｍ，４Ｈ）２．１９－２．３９（ｍ，３Ｈ）２．４０－２．５２（ｍ，２Ｈ）２．６３（ｄ，Ｊ＝９．２７Ｈｚ，１Ｈ）２．８０（ｓ，３Ｈ）２．９５（ｄｄ，Ｊ＝６．３４，４．３９Ｈｚ，１Ｈ）３．０６－３．１５（ｍ，１Ｈ）３．３１－３．４０（ｍ，２Ｈ）３．５０（ｓ，３Ｈ）３．６５－３．７２（ｍ，７Ｈ）３．７３－３．８０（ｍ，２Ｈ）４．１０－４．２４（ｍ，１Ｈ）５．４７（ｄｄ，Ｊ＝１５．１２，９．２７Ｈｚ，１Ｈ）５．８９（ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）６．２８（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． 1.7.5 H24 synthesis

General procedure 1.7 was used to give H24 as a colorless amorphous product (12.1 mg, 94% yield). LC / MS (ESI, m / z), 693.72 [M + H] ⁺ .
1H NMR (400MHz, methanol-d4) δ ppm 0.66 (d, J = 6.34Hz, 3H) 0.80 (d, J = 6.83Hz, 3H) 1.02 (d, J = 6.83Hz) , 4H) 1.08 (d, J = 6.34Hz, 4H) 1.32-1.37 (m, 8H) 1.44-1.50 (m, 4H) 1.65-1.68 (m) , 5H) 1.75-1.96 (m, 4H) 2.19-2.39 (m, 3H) 2.40-2.52 (m, 2H) 2.63 (d, J = 9.27Hz) , 1H) 2.80 (s, 3H) 2.95 (dd, J = 6.34, 4.39Hz, 1H) 3.06-3.15 (m, 1H) 3.31-3.40 (m) , 2H) 3.50 (s, 3H) 3.65-3.72 (m, 7H) 3.73-3.80 (m, 2H) 4.10-4.24 (m, 1H) 5.47 (Dd, J = 15.12, 9.27Hz, 1H) 5.89 (d, J = 10.24Hz, 1H) 6.28 (dd, J = 15.12, 10.73Hz, 1H).

一般的手順１．７を用いてＨ１８を無色のアモルファス生成物として得た（８．１ｍｇ、５３％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６７８．６８［Ｍ－Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６７（ｂｒ ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８７（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０３（ｂｒ ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１５－１．２４（ｍ，５Ｈ）１．２８（ｓ，３Ｈ）１．５０－１．６７（ｍ，５Ｈ）１．７５（ｂｒ ｓ，１Ｈ）１．８２－１．９４（ｍ，３Ｈ）２．３７－２．４４（ｍ，３Ｈ）２．５６－２．６０（ｍ，４Ｈ）２．７１－２．９３（ｍ，３Ｈ）３．００（ｂｒ ｔ，Ｊ＝５．１２Ｈｚ，１Ｈ）３．３７（ｂｒ ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）３．５３（ｓ，３Ｈ）３．５５－３．６３（ｍ，１Ｈ）３．６５－３．７６（ｍ，３Ｈ）３．８３－３．９１（ｍ，１Ｈ）４．０４（ｂｒ ｄ，Ｊ＝４．３９Ｈｚ，１Ｈ）４．０８－４．１６（ｍ，１Ｈ）４．４１－４．５３（ｍ，２Ｈ）５．４２－５．５５（ｍ，１Ｈ）５．９１（ｂｒ ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．１４－６．２８（ｍ，１Ｈ）７．２１（ｂｒ ｄ，Ｊ＝７．３２Ｈｚ，１Ｈ）． 1.7.6 Synthesis of H18

General procedure 1.7 was used to give H18 as a colorless amorphous product (8.1 mg, 53% yield). LC / MS (ESI, m / z), 678.68 [MH] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.67 (br d, J = 6.34Hz, 3H) 0.87 (d, J = 6.83Hz, 3H) 1.03 (br d, J = 6) .83Hz, 3H) 1.15-1.24 (m, 5H) 1.28 (s, 3H) 1.50-1.67 (m, 5H) 1.75 (br s, 1H) 1.82- 1.94 (m, 3H) 2.37-2.44 (m, 3H) 2.56-2.60 (m, 4H) 2.71-2.93 (m, 3H) 3.00 (br t) , J = 5.12Hz, 1H) 3.37 (br d, J = 10.24Hz, 1H) 3.53 (s, 3H) 3.55-3.63 (m, 1H) 3.65-3. 76 (m, 3H) 3.83-3.91 (m, 1H) 4.04 (br d, J = 4.39Hz, 1H) 4.08-4.16 (m, 1H) 4.41-4 .53 (m, 2H) 5.42-5.55 (m, 1H) 5.91 (br d, J = 10.73Hz, 1H) 6.14-6.28 (m, 1H) 7.21 ( br d, J = 7.32Hz, 1H).

一般的手順１．７を用いてＨ１３を無色のアモルファス生成物として得た（６．７ｍｇ、３６．７％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６５２．８６［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ４）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８１（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０２（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．０５－１．１０（ｍ，３Ｈ）１．１４－１．２３（ｍ，２Ｈ）１．２６（ｓ，４Ｈ）１．３４－１．５２（ｍ，２Ｈ）１．５２－１．５９（ｍ，１Ｈ）１．６５（ｂｒ ｓ，１Ｈ）１．７０（ｓ，４Ｈ）１．８２－１．９２（ｍ，２Ｈ）２．４１（ｑｄ，Ｊ＝１４．３９，６．１０Ｈｚ，３Ｈ）２．６３（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）２．７３（ｂｒ ｄ，Ｊ＝４．３９Ｈｚ，３Ｈ）２．９５（ｂｒ ｔ，Ｊ＝５．１２Ｈｚ，２Ｈ）２．９９－３．０９（ｍ，３Ｈ）３．３０－３．３６（ｍ，１Ｈ）３．５０（ｓ，３Ｈ）３．５３－３．６５（ｍ，２Ｈ）３．６５－３．７８（ｍ，４Ｈ）４．２４－４．４２（ｍ，２Ｈ）４．５５（ｂｒ ｓ，１Ｈ）５．３６－５．５５（ｍ，１Ｈ）５．８９（ｂｒ ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２１－６．４３（ｍ，１Ｈ）． 1.7.7 Synthesis of H13

General procedure 1.7 was used to give H13 as a colorless amorphous product (6.7 mg, 36.7% yield). LC / MS (ESI, m / z), 652.86 [M + H] ⁺ .
1H NMR (400MHz, methanol-d4) δ ppm 0.66 (d, J = 6.34Hz, 3H) 0.81 (d, J = 6.34Hz, 3H) 1.02 (d, J = 6.34Hz) , 3H) 1.05-1.10 (m, 3H) 1.14-1.23 (m, 2H) 1.26 (s, 4H) 1.34-1.52 (m, 2H) 1.52 -1.59 (m, 1H) 1.65 (br s, 1H) 1.70 (s, 4H) 1.82-1.92 (m, 2H) 2.41 (qd, J = 14.39, 6.10Hz, 3H) 2.63 (d, J = 9.76Hz, 1H) 2.73 (br d, J = 4.39Hz, 3H) 2.95 (br t, J = 5.12Hz, 2H) 2.99-3.09 (m, 3H) 3.30-3.36 (m, 1H) 3.50 (s, 3H) 3.53-3.65 (m, 2H) 3.65-3. 78 (m, 4H) 4.24-4.42 (m, 2H) 4.55 (br s, 1H) 5.36-5.55 (m, 1H) 5.89 (br d, J = 10. 73Hz, 1H) 6.21-6.43 (m, 1H).

一般的手順１．７を用いてＨ１４を無色のアモルファス固体として得た（１５．５ｍｇ、８３％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６６６．９０［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６５（ｂｒ ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｂｒ ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０２（ｂｒ ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１４－１．２８（ｍ，８Ｈ）１．３８－１．５７（ｍ，２Ｈ）１．５７－１．６５（ｍ，２Ｈ）１．８１－１．８９（ｍ，２Ｈ）２．０４－２．１９（ｍ，２Ｈ）２．４１（ｂｒ ｄ，Ｊ＝４．８８Ｈｚ，３Ｈ）２．５４（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）２．６５（ｓ，３Ｈ）２．９１－３．０３（ｍ，５Ｈ）３．３３（ｂｒ ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．５２（ｓ，３Ｈ）３．７０（ｂｒ ｓ，５Ｈ）３．８１－３．８７（ｍ，１Ｈ）４．０８－４．１８（ｍ，２Ｈ）４．５２（ｂｒ ｄ，Ｊ＝６．３４Ｈｚ，１Ｈ）５．４５（ｂｒ ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８９（ｂｒ ｄ，Ｊ＝１０．７３Ｈｚ，１Ｈ）６．２１（ｂｒ ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，２Ｈ）７．１５（ｂｒ ｄ，Ｊ＝７．３２Ｈｚ，１Ｈ）． 1.7.8 Synthesis of H14

H14 was obtained as a colorless amorphous solid using general procedure 1.7 (15.5 mg, 83% yield). LC / MS (ESI, m / z), 666.90 [M + H] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.65 (br d, J = 6.34Hz, 3H) 0.85 (br d, J = 6.83Hz, 3H) 1.02 (br d, J = 6.34Hz, 3H) 1.14-1.28 (m, 8H) 1.38-1.57 (m, 2H) 1.57-1.65 (m, 2H) 1.81-1.89 ( m, 2H) 2.04-2.19 (m, 2H) 2.41 (br d, J = 4.88Hz, 3H) 2.54 (d, J = 9.76Hz, 1H) 2.65 (s) , 3H) 2.91-3.03 (m, 5H) 3.33 (br d, J = 9.76Hz, 1H) 3.52 (s, 3H) 3.70 (br s, 5H) 3.81 -3.87 (m, 1H) 4.08-4.18 (m, 2H) 4.52 (br d, J = 6.34Hz, 1H) 5.45 (br dd, J = 15.12, 8) .78Hz, 1H) 5.89 (br d, J = 10.73Hz, 1H) 6.21 (br dd, J = 14.88, 10.98Hz, 2H) 7.15 (br d, J = 7. 32Hz, 1H).

一般的手順１．７を用いてＨ１７を無色のアモルファス固体として得た（１２．９４ｍｇ、６８％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６８０．６６［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ４）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．８３Ｈｚ，４Ｈ）０．８１（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）０．９７－１．０４（ｍ，５Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，５Ｈ）１．１０－１．１６（ｍ，２Ｈ）１．２０－１．２３（ｍ，１Ｈ）１．２５（ｓ，４Ｈ）１．６９（ｄ，Ｊ＝０．９８Ｈｚ，４Ｈ）１．８３（ｂｒ ｄ，Ｊ＝３．４１Ｈｚ，１Ｈ）１．８７（ｂｒ ｄ，Ｊ＝４．３９Ｈｚ，１Ｈ）１．９０（ｂｒ ｄ，Ｊ＝４．３９Ｈｚ，１Ｈ）２．２４－２．３０（ｍ，１Ｈ）２．４０（ｂｒ ｄｄ，Ｊ＝１３．９０，９．０２Ｈｚ，２Ｈ）２．５８－２．６２（ｍ，５Ｈ）２．６４（ｓ，１Ｈ）２．８４（ｂｒ ｔ，Ｊ＝４．８８Ｈｚ，５Ｈ）２．９５（ｄｄ，Ｊ＝６．３４，４．３９Ｈｚ，１Ｈ）３．３０－３．３２（ｍ，１Ｈ）３．５０（ｓ，３Ｈ）３．５６－３．６４（ｍ，６Ｈ）３．７４－３．７９（ｍ，１Ｈ）４．０２（ｂｒ ｔ，Ｊ＝５．３７Ｈｚ，３Ｈ）４．３２－４．３５（ｍ，１Ｈ）４．３５－４．３７（ｍ，１Ｈ）５．４５（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．８５－５．９１（ｍ，１Ｈ）６．２７（ｄｄ，Ｊ＝１５．１２，１０．７３Ｈｚ，１Ｈ）． 1.7.9 Synthesis of H17

H17 was obtained as a colorless amorphous solid using general procedure 1.7 (12.94 mg, 68% yield). LC / MS (ESI, m / z), 680.66 [M + H] ⁺ .
1H NMR (400MHz, methanol-d4) δ ppm 0.66 (d, J = 6.83Hz, 4H) 0.81 (d, J = 7.32Hz, 3H) 0.97-1.04 (m, 5H) ) 1.08 (d, J = 6.34Hz, 5H) 1.10-1.16 (m, 2H) 1.20-1.23 (m, 1H) 1.25 (s, 4H) 1.69 (D, J = 0.98Hz, 4H) 1.83 (br d, J = 3.41Hz, 1H) 1.87 (br d, J = 4.39Hz, 1H) 1.90 (br d, J = 4.39Hz, 1H) 2.24-2.30 (m, 1H) 2.40 (br dd, J = 13.90, 9.02Hz, 2H) 2.58-2.62 (m, 5H) 2 .64 (s, 1H) 2.84 (br t, J = 4.88Hz, 5H) 2.95 (dd, J = 6.34, 4.39Hz, 1H) 3.30-3.32 (m, 1H) 3.50 (s, 3H) 3.56-3.64 (m, 6H) 3.74-3.79 (m, 1H) 4.02 (br t, J = 5.37Hz, 3H) 4 .32-4.35 (m, 1H) 4.35-4.37 (m, 1H) 5.45 (dd, J = 15.12, 8.78Hz, 1H) 5.85-5.91 (m) , 1H) 6.27 (dd, J = 15.12, 10.73Hz, 1H).

一般的手順１．７を用いてＨ１９を無色のアモルファス固体として得た（５．５３ｍｇ、５６．４％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６９４．０８［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，メタノール－ｄ４）δ ｐｐｍ ０．６６（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．８１（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）０．９５－０．９５（ｍ，１Ｈ）１．０２（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０８（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１２－１．２２（ｍ，２Ｈ）１．２６（ｓ，４Ｈ）１．３４－１．５２（ｍ，７Ｈ）１．６２（ｂｒ ｄ，Ｊ＝１３．６６Ｈｚ，２Ｈ）１．６８（ｓ，４Ｈ）１．８１－１．９３（ｍ，３Ｈ）２．０８－２．０８（ｍ，１Ｈ）２．２０－２．３９（ｍ，２Ｈ）２．３９－２．５０（ｍ，１Ｈ）２．６１－２．６８（ｍ，４Ｈ）２．９０－２．９７（ｍ，５Ｈ）３．１１－３．１５（ｍ，２Ｈ）３．３０－３．３４（ｍ，１Ｈ）３．５０（ｓ，３Ｈ）３．５６－３．６２（ｍ，４Ｈ）３．７６（ｔ，Ｊ＝６．３４Ｈｚ，１Ｈ）４．１１－４．２２（ｍ，１Ｈ）５．４５（ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．９０（ｂｒ ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．２９（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）． 1.7.10 Synthesis of H19

H19 was obtained as a colorless amorphous solid using general procedure 1.7 (5.53 mg, 56.4% yield). LC / MS (ESI, m / z), 694.08 [M + H] ⁺ .
1H NMR (400MHz, methanol-d4) δ ppm 0.66 (d, J = 6.83Hz, 3H) 0.81 (d, J = 6.83Hz, 3H) 0.95-0.95 (m, 1H) ) 1.02 (d, J = 6.83Hz, 3H) 1.08 (d, J = 6.34Hz, 3H) 1.12-1.22 (m, 2H) 1.26 (s, 4H) 1 .34-1.52 (m, 7H) 1.62 (br d, J = 13.66Hz, 2H) 1.68 (s, 4H) 1.81-1.93 (m, 3H) 2.08- 2.08 (m, 1H) 2.20-2.39 (m, 2H) 2.39-2.50 (m, 1H) 2.61-2.68 (m, 4H) 2.90-2. 97 (m, 5H) 3.11-3.15 (m, 2H) 3.30-3.34 (m, 1H) 3.50 (s, 3H) 3.56-3.62 (m, 4H) 3.76 (t, J = 6.34Hz, 1H) 4.11-4.22 (m, 1H) 5.45 (dd, J = 15.12, 8.78Hz, 1H) 5.90 (br d) , J = 11.22Hz, 1H) 6.29 (dd, J = 14.88, 10.98Hz, 1H).

一般的手順１．７を用いてＨ２１を得て、これは無色のアモルファス固体として得られた（１２．３ｍｇ、６２．１％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），７０８．０３［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６７（ｂｒ ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）０．８５（ｂｒ ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０２（ｂｒ ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１５－１．２９（ｍ，９Ｈ）１．２９－１．４２（ｍ，３Ｈ）１．４７－１．６３（ｍ，６Ｈ）１．７４－１．７９（ｍ，１Ｈ）１．８６（ｂｒ ｄｄ，Ｊ＝１３．１７，３．９０Ｈｚ，２Ｈ）２．４０（ｂｒ ｄ，Ｊ＝５．３７Ｈｚ，３Ｈ）２．５１－２．５５（ｍ，４Ｈ）２．７８（ｂｒ ｓ，４Ｈ）２．９６（ｂｒ ｔ，Ｊ＝５．１２Ｈｚ，１Ｈ）３．１８（ｔｄ，Ｊ＝１２．０７，６．１０Ｈｚ，２Ｈ）３．３７（ｂｒ ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．５２（ｓ，３Ｈ）３．５７（ｂｒ ｓ，４Ｈ）３．６９（ｓ，４Ｈ）３．８４（ｂｒ ｔ，Ｊ＝５．８５Ｈｚ，１Ｈ）４．５０－４．５７（ｍ，１Ｈ）４．８５－４．９３（ｍ，２Ｈ）５．４４（ｂｒ ｄｄ，Ｊ＝１５．１２，８．７８Ｈｚ，１Ｈ）５．９２（ｂｒ ｄ，Ｊ＝１０．２４Ｈｚ，１Ｈ）６．１５－６．３８（ｍ，１Ｈ）７．２４－７．２９（ｍ，２Ｈ）８．２４（ｂｒ ｓ，１Ｈ）． 1.7.11 Synthesis of H21

H21 was obtained using the general procedure 1.7, which was obtained as a colorless amorphous solid (12.3 mg, 62.1% yield). LC / MS (ESI, m / z), 708.03 [M + H] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.67 (br d, J = 6.34Hz, 3H) 0.85 (br d, J = 6.83Hz, 3H) 1.02 (br d, J = 6.83Hz, 3H) 1.15-1.29 (m, 9H) 1.29-1.42 (m, 3H) 1.47-1.63 (m, 6H) 1.74-1.79 ( m, 1H) 1.86 (br dd, J = 13.17, 3.90Hz, 2H) 2.40 (br d, J = 5.37Hz, 3H) 2.51-2.55 (m, 4H) 2.78 (br s, 4H) 2.96 (br t, J = 5.12Hz, 1H) 3.18 (td, J = 12.07, 6.10Hz, 2H) 3.37 (br d, J) = 9.76Hz, 1H) 3.52 (s, 3H) 3.57 (br s, 4H) 3.69 (s, 4H) 3.84 (br t, J = 5.85Hz, 1H) 4.50 -4.57 (m, 1H) 4.85-4.93 (m, 2H) 5.44 (br dd, J = 15.12, 8.78Hz, 1H) 5.92 (br d, J = 10) .24Hz, 1H) 6.15-6.38 (m, 1H) 7.24-7.29 (m, 2H) 8.24 (br s, 1H).

ステップ１：ジクロロメタン（２ｍｌ）中の（（２Ｒ，５Ｓ，６Ｓ）－６－（（Ｓ，２Ｅ，４Ｅ）－７－（（２Ｒ，３Ｒ）－３－（（２Ｒ，３Ｒ，４Ｒ）－３－メトキシ－４－（（トリエチルシリル）オキシ）ペンタン－２－イル）－２－メチルオキシラン－２－イル）－６－メチルヘプタ－２，４－ジエン－２－イル）－５－メチルテトラヒドロ－２Ｈ－ピラン－２－ｌ）メタンアミン（１６．６ｍｇ、０．０２７ｍｍｏｌ）及び４－カルボキシ－１－シクロヘキサンメタノール（シス・トランス混合物、５．１１ｍｇ、０．０３２ｍｍｏｌ）の溶液に室温でＥＤＣ（６．２０ｍｇ、０．０３２ｍｍｏｌ）及びＨＯＢＴ（４．５４ｍｇ、０．０３ｍｍｏｌ）を加えた。この反応混合物を室温で１６時間撹拌した。次に、この混合物をジクロロメタンで希釈し、有機層を水及びブラインで洗浄し、次に無水硫酸ナトリウムで乾燥させた。固体をろ過し、ろ液を濃縮し、シリカゲルクロマトグラフィーにより精製して、所望の生成物を無色の油として得た（８．３ｍｇ、４７％収率） 1.8 Synthesis of H22 and H25 H22 and H25 were prepared by the following general procedure.

Step 1: ((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R) -3-((2R, 3R, 4R) -3) in dichloromethane (2 ml)) -Methoxy-4- ((triethylsilyl) oxy) pentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H -Pyran-2-l) EDC (6.20 mg) at room temperature in a solution of methaneamine (16.6 mg, 0.027 mmol) and 4-carboxy-1-cyclohexanemethanol (cis-trans mixture, 5.11 mg, 0.032 mmol). , 0.032 mmol) and HOBT (4.54 mg, 0.03 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. The mixture was then diluted with dichloromethane and the organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. The solid was filtered, the filtrate was concentrated and purified by silica gel chromatography to give the desired product as a colorless oil (8.3 mg, 47% yield).

Step 2: 4- (Hydroxymethyl) -N-(((2R, 5S, 6S) -6-((S, 2E, 4E) -7-((2R, 3R)-) in dichloromethane (1 ml) at room temperature 3-((2R, 3R, 4R) -3-methoxy-4-((triethylsilyl) oxy) pentan-2-yl) -2-methyloxylan-2-yl) -6-methylhepta-2,4-diene -2-Il) -5-Methyltetrahydro-2H-Pyran-2-yl) Methyl) Cyclohexanecarboxamide (21 mg, 0.032 mmol) and DIPEA (0.028 ml, 0.158 mmol) in a cis, trans mixture solution. 4-Nitrophenyl nochloride (12.75 mg, 0.063 mmol) and DMAP (1.932 mg, 0.016 mmol) were added in small portions. The resulting mixture was stirred at the same temperature for 16 hours. Next, 1-methylpiperazine (31.7 mg, 0.316 mmol) was added, and the mixture was further stirred for 1 hour. The reaction mixture was diluted with dichloromethane and the organic layer was washed with water and brine and then dried over Na ₂ SO ₄ . The solid was filtered and the filtrate was concentrated to dryness. The obtained residue was dissolved in 1.0 mL of MeOH and 10 mg of p-TsOH and stirred at room temperature for 2 hours. The reaction was quenched by adding 100 uL of DIPEA and then the solvent was removed in vacuo. The obtained residue was purified by NH-silica gel chromatography (Hep / AcOEt = 50/50 to 0/100) to obtain H25 and H22.

Ｈ２５は無色のアモルファス生成物として得られた（７．０ｍｇ、３２．７％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６７６．９４［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６８（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０２（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）１．１０（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１７（ｄ，Ｊ＝６．３４Ｈｚ，４Ｈ）１．２５（ｓ，３Ｈ）１．４７－１．６２（ｍ，１１Ｈ）１．７１（ｓ，３Ｈ）１．８０－１．８６（ｍ，４Ｈ）２．１３（ｓ，１Ｈ）２．２７－２．３６（ｍ，１０Ｈ）２．４９－２．６５（ｍ，１Ｈ）２．９９－３．１２（ｍ，１Ｈ）３．２９（ｄ，Ｊ＝１０．２０Ｈｚ，１Ｈ）３．３２（ｓ，４Ｈ）３．４７（ｂｒ ｔ，Ｊ＝４．８８Ｈｚ，４Ｈ）３．７２－３．７３（ｍ，１Ｈ）３．７３－３．７４（ｍ，１Ｈ）３．９９（ｄ，Ｊ＝６．８３Ｈｚ，２Ｈ）４．２１－４．３６（ｍ，１Ｈ）５．５６－５．７１（ｍ，１Ｈ）５．９２（ｓ，１Ｈ）６．１８－６．３１（ｍ，１Ｈ）． 1.8.1 Synthesis of H25

H25 was obtained as a colorless amorphous product (7.0 mg, 32.7% yield). LC / MS (ESI, m / z), 676.94 [M + H] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.68 (d, J = 6.83Hz, 3H) 1.02 (d, J = 7.32Hz, 3H) 1.10 (d, J = 6.83Hz) , 3H) 1.17 (d, J = 6.34Hz, 4H) 1.25 (s, 3H) 1.47-1.62 (m, 11H) 1.71 (s, 3H) 1.80-1 .86 (m, 4H) 2.13 (s, 1H) 2.27-2.36 (m, 10H) 2.49-2.65 (m, 1H) 2.99-3.12 (m, 1H) ) 3.29 (d, J = 10.20Hz, 1H) 3.32 (s, 4H) 3.47 (br t, J = 4.88Hz, 4H) 3.72-3.73 (m, 1H) 3.73-3.74 (m, 1H) 3.99 (d, J = 6.83Hz, 2H) 4.21-4.36 (m, 1H) 5.56-5.71 (m, 1H) 5.92 (s, 1H) 6.18-6.31 (m, 1H).

Ｈ２２は無色のアモルファス生成物として得られた（５．６ｍｇ、２６．２％収率）。ＬＣ／ＭＳ（ＥＳＩ，ｍ／ｚ），６７６．８９［Ｍ＋Ｈ］^＋．
１Ｈ ＮＭＲ（４００ＭＨｚ，クロロホルム－ｄ）δ ｐｐｍ ０．６８（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．０２（ｄ，Ｊ＝６．８３Ｈｚ，３Ｈ）１．１０（ｄ，Ｊ＝７．３２Ｈｚ，３Ｈ）１．１４（ｄ，Ｊ＝６．３４Ｈｚ，３Ｈ）１．１７（ｓ，３Ｈ）１．２１－１．４１（ｍ，２Ｈ）１．４９－１．５５（ｍ，５Ｈ）１．５８－１．６２（ｍ，６Ｈ）１．７０（ｓ，３Ｈ）１．８０－１．８６（ｍ，４Ｈ）２．２６－２．３０（ｍ，４Ｈ）２．３５（ｂｒ ｓ，４Ｈ）２．４６－２．５７（ｍ，１Ｈ）２．８９（ｄｄ，Ｊ＝３．６６，２．２０Ｈｚ，１Ｈ）３．０１－３．１１（ｍ，１Ｈ）３．２７（ｄ，Ｊ＝９．７６Ｈｚ，１Ｈ）３．３８（ｓ，３Ｈ）３．４７（ｂｒ ｔ，Ｊ＝４．８８Ｈｚ，４Ｈ）３．５３（ｄｄｄ，Ｊ＝１０．２４，６．８３，３．４１Ｈｚ，１Ｈ）３．７６（ｄ，Ｊ＝５．３７Ｈｚ，１Ｈ）３．８９（ｄｄ，Ｊ＝６．３４，１．９５Ｈｚ，１Ｈ）３．９９（ｄ，Ｊ＝７．３２Ｈｚ，２Ｈ）５．６８－５．７７（ｍ，１Ｈ）５．９１（ｄ，Ｊ＝１１．２２Ｈｚ，１Ｈ）６．１８（ｄｄ，Ｊ＝１４．８８，１０．９８Ｈｚ，１Ｈ）． 1.8.2 Synthesis of H22

H22 was obtained as a colorless amorphous product (5.6 mg, 26.2% yield). LC / MS (ESI, m / z), 676.89 [M + H] ⁺ .
1H NMR (400MHz, chloroform-d) δ ppm 0.68 (d, J = 6.83Hz, 3H) 1.02 (d, J = 6.83Hz, 3H) 1.10 (d, J = 7.32Hz) , 3H) 1.14 (d, J = 6.34Hz, 3H) 1.17 (s, 3H) 1.21-1.41 (m, 2H) 1.49-1.55 (m, 5H) 1 .58-1.62 (m, 6H) 1.70 (s, 3H) 1.80-1.86 (m, 4H) 2.26-2.30 (m, 4H) 2.35 (br s, 4H) 2.46-2.57 (m, 1H) 2.89 (dd, J = 3.66, 2.20Hz, 1H) 3.01-3.11 (m, 1H) 3.27 (d, J = 9.76Hz, 1H) 3.38 (s, 3H) 3.47 (br t, J = 4.88Hz, 4H) 3.53 (ddd, J = 10.24,6.83, 3.41Hz , 1H) 3.76 (d, J = 5.37Hz, 1H) 3.89 (dd, J = 6.34, 1.95Hz, 1H) 3.99 (d, J = 7.32Hz, 2H) 5 .68-5.77 (m, 1H) 5.91 (d, J = 11.22Hz, 1H) 6.18 (dd, J = 14.88, 10.98Hz, 1H).

Example 2
An exemplary harboxydiene spliceosome regulator payload used in the preparation of ADCs was profiled. The payload was evaluated for binding to the SF3b complex, in vitro splicing activity, and ability to inhibit cell growth.

2.1 In vitro splicing (IVS)
In vitro splicing assays were performed to assess payload activity in cell-free systems. The payload was incubated with nuclear extract and premRNA substrate minigene.

HeLa nuclear extract preparation: HeLa S3 cell pellet is resuspended in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl ₂ , 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT) and suspended. Was adjusted to a total of 5 pack cell volume (PCV). After centrifugation, the supernatant was discarded and the cells were incubated with hypotonic buffer at 3PCV for 10 minutes on ice. Cells were lysed using a Downs homogenizer and then centrifuged. Discard the supernatant and use a 1/2 pack nuclear volume (PNV) low salinity buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl ₂ , 20 mM KCl, 0.2 mM EDTA, 25% glycerol, The pellet was resuspended in 0.2 mM PMSF, 0.5 mM DTT) followed by 1 / 2PNV high salt buffer (same as low salt buffer except 1.4 M KCl). The nuclei were gently mixed for 30 minutes and then centrifuged. The supernatant (nuclear extract) was then dialyzed against storage buffer (20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.2 mM PMSF, 0.5 mM DTT). The protein concentration was determined using a NanoDrop 8000 UV-Visible Spectrophotometer (Thermo Fisher Scientific).

IVS: Cloning all Ad2-derived sequences (Pellizzoni et al. (1998) Cell 95 (5): 615-24) into pcDNA3.1 (+) vector (Promega) using 5'EcoRI and 3'XbaI restriction sites. did. These plasmids were linearized with XbaI and used as a DNA template for in vitro transcription reactions. The FtzΔi-free intron plasmid (Luo and Red (1999) 96 (26): 14937-42) was linearized using EcoRI. All RNA was transcribed in vitro and then purified using the MEGAScript T7 (Invitrogen) and MegaClear (Invitrogen) kits, respectively. For splicing reactions using Ad2 variant pre-mRNA, 1 μL reaction was prepared using 8 μg nuclear extract prepared from HeLa S3, 2 ng pre-mRNA, 0.2 ng FTZΔi, and various concentrations of compounds or DMSO. .. After pre-incubating at 30 ° C. for 15 minutes, 1 μL splicing activation buffer (0.5 mM ATP, 20 mM creatine phosphate, 1.6 mM MgCl ₂ ) was added and the reaction was incubated at 30 ° C. for 90 minutes. The reaction was then quenched with 13 μL DMSO and 25 nL was used for RT-qPCR. _{TaqMan RNA-to-C T 1} -step kit (Life Technologies), RNA from splicing reaction, Ad2 (Forward: ACTCTCTTCCGCATCGCTGT; Reverse: CCGACGGGTTTCCGATCCAA; Probe: CTGTTGGGCTCGCGGTTG) and Ftz (Forward: TGGCATCAGATTGCAAAGAC; Reverse: ACGCCGGGTGATGTATCTAT; Probe: CGAAACGCACCCGTCAGACG ) An RT-qPCR reaction was prepared using the mRNA primer-probe set. Prism 7 (Graphpad) was used for the non-linear regression curve fitting of the splicing products formed and normalized to control (DMSO) samples.

Given that all payloads tested specifically bind to the SF3b complex and demonstrate similar binding profiles, it was hypothesized that all payloads should also regulate splicing to the same extent. All payloads significantly regulated the splicing of Ad2.2 premRNA (see Table 13). In the presence of the payload, a decrease in splice production was observed.

2.2 Cell viability HCC1954 (American Type Culture Collection (ATCC)) Tissue culture of ductal carcinoma cells in a flat-bottomed 96-well tissue culture plate (Corning) supplemented with 10% Hermo Fisher Scientific tissue culture. The medium was plated with 2000 cells / well. Cells were treated with a 3-fold serial dilution of compound from 200 nM to 0.03 nΜ. Each concentration was tested in triplicate. At the time of treatment, the CellTiter-Glo® 2.0 luminescent cell viability assay was used according to the manufacturer's recommendations (Promega; # G9241) to evaluate plates of untreated cells. CellTiter-Glo® 2.0 reagent was added to the medium, incubated and assayed on the EnVision Multilabel Reader (PerkinElmer). The value represents the time point 0 (T0). The number of viable cells after 144 hours of compound treatment (T144) was also determined using the CellTiter-Glo® 2.0 luminescent cell viability assay. Growth rates at each compound concentration level were calculated using emission values at time point 0 (T0), DMSO control growth (C), and test growth (T144) in the presence of the compound. The growth inhibition rate was calculated as [(T144-T0) / (C—T0)] × 100 for the concentration of T144 ≧ T0 or [(T144-T0) / T0] × 100 for the concentration of T144 <T0. Prism 7 (Graphpad) and non-linear regression analysis and log (inhibitor) vs. A dose-response curve plot was created using a fitting with response-variable slope (4 parameters).

Cell viability dose responses were determined in HER2-amplified HCC1954 breast cancer cells for all payloads. Most of the payloads tested exhibited GI ₅₀ values in the low nanomolar concentration range (ie, the concentration of the compound to cause a 50% reduction in cell proliferation) (see Table 13).

Example 3
The exemplary payload compound evaluated in Example 2 was conjugated to an exemplary anti-HER2 antibody (trastuzumab) via the cysteine residue of the antibody. The preparation and evaluation of exemplary anti-HER2 ADCs are described below.

3.1 Antibodies For the preparation of anti-HER2 ADCs (also referred to herein as SMLA), trastuzumab antibodies (“AB185”) (Molina et al. (2001) Cancer Res. 61 (12): 4744-9). It was used.

3.2 Bioconjugation 10 mg / mL antibody (trastuzumab) in PBS buffer (pH 7.0) mixed with 5 mM TCEP (2-4 mol equivalents) (Thermo Fisher Scientific; # 77720) to disrupt interchain disulfide bonds. did. The reaction was gently mixed at 22 ° C. for 3 hours. Propylene glycol (15% v / v) was then added, followed by 8 molar equivalents of the linker-payload (6 mM stock in DMSO) and the solutions were mixed well. The reaction was placed on a rotating plate in a 22 ° C. incubator. After 2 hours of conjugation, the reaction mixture was purified and the unconjugated payload was placed in DPBS (pH 7.5) in AKTA GE M150 (HiTrap ™ 26/10 desalting column; flow rate: 3 mL / min). Removed by (GE Healthcare Bio-Sciences). The resulting conjugate was concentrated by Amicon ultrafiltration (30 kDa, Ultra-4) (EMD Millipore) and sterilized by a 0.22 μm PVDF disposable filter (EMD Millipore). The final clear solution was measured by UV-VIS and the antibody concentration ([mAb]; mol / L) and the conjugated payload concentration ([LD]; mol / L) were determined by Lambert-Beer's law (A =). E · c · l) and determined by the following formula:
A _280nm = E ^mAb _280nm × [mAb] × l + E ^LD _280nm × [LD] × l
A _252nm = E ^mAb _252nm × [mAb] × l + E ^LD _252nm × [LD] × l
E ^mAb _{280 nm} : trastuzumab = 213,380 cm ^-1 M ^-1
E ^mAb _{252 nm} : trastuzumab = 79,112 cm ^-1 M ^-1
E ^LD _280nm = 800cm ^-1 M ^-1
E ^LD _252nm = 31,000cm ^-1 M ^-1
Abbreviation: c-molar concentration; l-optical path length (Nanodrop: 0.1 cm); E-molar extinction coefficient; A-absorbance.

3.3 Biophysical characterization For an exemplary anti-HER2 ADC, Harbo by liquid chromatography mass spectrometry (LC / MS), size exclusion chromatography (SEC), and reverse phase high performance liquid chromatography (HPLC), respectively. The xidien splicing regulator-to-antibody ratio (HAR), aggregation rate, and non-conjugated payload rate were analyzed. Generally, the conjugate contained less than 2% free drug and less than 10% aggregation.

33.1 LC / MS Analysis-HAR
LC / MS analysis was performed using an Agilent 1290 UPLC system coupled to an Agilent G6224A Accurate Mass TOF mass spectrometer. The conjugate was deglycosylated at 37 ° C. for 4 hours with PNGase F (New England Biolabs; # P0705L), denatured with 8M Gdn-HCl (Sigma; # G9284), and finally DTT (5 mM final concentration) (Promega; #). V3151) was used to separate the light chain domain and the heavy chain domain. The prepared sample was injected into an Agilent PLRP-S column (2.1 × 150 mm, 8 μm) and eluted with a 25% B-50% B gradient at room temperature (RT) over 28 minutes. The mobile phase A was 0.05% TFA-containing water, the mobile phase B was 0.04% TFA-containing acetonitrile, and the flow rate was 1 mL / min. HAR was calculated from the deconvolution mass spectra by weighted averaging the intensities of the unconjugated and drug conjugated peaks for the light chain (L0 or L1) and heavy chain (H0, H1, H2, and H3). Formula: (HAR _LC x 2) + (HAR _HC x 2) = total HAR was used to calculate the total DA of intact conjugates. HAR values for exemplary anti-HER2 ADCs are reported in Table 12.

3.3.2 SEC Analysis-Aggregation Using a TOSON-G3000SWXL (# 008541) column, in 0.2 M Potassium Phosphate (pH 7) containing 0.25 mM Potassium Chloride and 15% (v / v) IPA, Size exclusion chromatography was performed at a flow rate of 0.75 mL / min. For the high molecular weight monomer conjugate component, the peak area absorbance at 280 nm was determined by under-curve surface integral. The monomeric proportions of the exemplary anti-HER2 ADC are reported in Table 12.

3.3.3 HPLC Analysis-Free Harboxy Diene Splicing Regulator The conjugate of interest was precipitated on ice with 10 volumes of acetonitrile for 2 hours and spun down. The supernatant containing the residual unconjugated payload is then injected into an Agilent Poroshell 120 SB-C18 120A column (4.6 x 100 mm, 2.7 μm) and 10 at room temperature with a 45% B-70% B gradient. It was eluted over a minute. Mobile phase A was 100% water, mobile phase B was 100% acetonitrile, and the flow rate was 0.6 mL / min, detected at 252 nm. UV detection quantified the amount of residual free harboxidien splicing regulator compared to the external calibration curve of the non-conjugated linker-payload. The rate of free harboxidiene splicing regulators of exemplary anti-HER2 ADCs is reported in Table 12.

3.4 Binding characterization 3.4.1 FACS binding to target-positive cells Binding to target-positive cells by non-conjugated anti-HER2 antibody and anti-HER2 ADC is evaluated by flow cytometry using indirect immunofluorescence. did. JIMT1 cells (DSMZ), a breast cancer cell line that endogenously expresses HER2, were plated on a V-bottom 96-well plate (Greener Bio-One) (5 × 10 ⁴ cells / well) and assay medium (10% (10%). w / v) Incubated at 4 ° C. for 2 hours with test compounds diluted to various concentrations in RPMI-1640) supplemented with fetal bovine serum albumin (Thermo Fisher Scientific). The cells were then washed with PBS + 2% FBS (FACS buffer) and stained with phycoerythrin-labeled (PE) goat anti-human immunoglobulin G (IgG) antibody (Invitrogen) at 4 ° C. in the dark for 40 minutes. Cells were washed with cold FACS buffer and fixed with FluroFix buffer (Biolegend) at room temperature for 30 minutes. The fixative was rinsed with FACS buffer. The immobilized cells were analyzed for geometric mean fluorescence of PE using the LSR Rotessa flow cytometer (BD Bioscience). Trastuzumab and T-DM1 (DM1 conjugated to trastuzumab) were included as controls.

3.5 In vitro analysis 3.5.1 Cell viability was tested on the ability of anti-HER2 ADCs to inhibit cell growth in several HER2 amplified cell lines. HCC 1954 (ATCC), 2000 cells / well) cell line was used. Cell viability analysis was performed as described in Section 2.3.

Surprisingly, not all ADCs were active in HCC1954 cells, despite similar binding profiles (see Table 13) and similar biochemical properties of the payload. For example, ADCs containing an ADL2 linker had a lower ability to inhibit cell growth (eg, compare the activities of ADL1-H1 and ADL2-H1).

3.5.2 Caco-2 permeable Caco-2 cells with ADC payload were cultured in transwell 24-well plates at 37 ° C., 95% humidity and 5% CO ₂ for 21 days. The integrity of the cell monolayer was confirmed by TEER (transepithelial electrical resistance) and Lucifer Yellow. The payload was duplicated on both sides of the cell monolayer and spiked separately at 10 μM. By collecting aliquots from both chambers immediately after treatment (t = 0) and after incubation for 2 hours, the apical-to-bottom-side (AB) direction and the bottom-to-top-to-top (B-) direction. The transmission speed in the A) direction was determined. Samples were protein precipitated in an organic solvent containing an internal standard and analyzed by LC-MS / MS (SCIEX; API 5500). Transparency (cm / sec) values were determined using payload / internal standard temporal area ratio reactions in both directions. The outward flux ratio was calculated by dividing BA / AB. The control compounds for low and high permeability as well as outward flux behaved as expected. The permeability values are reported in Table 12.

3.5.3 Chemical stability of ADC payload Harboxydiensplying regulator in Mcilvane (citrate-phosphate) buffer, pH 5.5 (Boston Bioproducts; # BB-2466) at a final concentration of 20 μM (# BB-2466). Incubated with less than 0.5% DMSO) from stock solution. Harboxydiene splicing regulator solutions and internal standards were pipetted onto 96-well plates, subjected to UPLC (Waters Fitness H-class) and analyzed for initial chromatographic signals (t = 0). The column was a Waters UPLC HSS T3 1.8 μm 2.1 × 50 mm column (# 186003538). A mobile phase A of 95% to 10% gradient was used over 1 minute, where A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile (flow 0.9 mL / min). ). The rest of the harboxydiene splicing regulator solution was retained on a plate shaker (Eppendorf ThermoMixer) at 37 ° C. Sample analysis with UPLC was repeated at 24, 72, and 96 hours after incubation at 37 ° C. The area ratio response of the harboxydiene splicing regulator and internal standard was determined at time 3: 0, day 1, and either day 3 or day 4. The time point 0 was set to 100. The area ratio reaction at a later time point was compared with the time point 0. The survival rate was calculated as follows: (area ratio X-day / area ratio 0 time point) × 100 = survival rate%. The slope of the line was calculated by comparing the logarithm of the% residual rate and the time point with Excel. The half-life was calculated by ln (2) / slope in Excel. Report to Table 12.

Example 4
Exemplary payload compounds were evaluated as described below.

4.1 In vitro analysis 4.1.1 Cell viability For exemplary payload compounds Cell viability dose responses were determined in HER2-amplified HCC1954 breast cancer cells and NCI-N87 gastric cancer cells.

HCC 1954 (American Type Culture Collection (ATCC)) breast duct cancer cells or NCI-N87 (ATCC) gastric cancer cells supplemented with 10% bovine fetal serum (Thermo Fisher Scientific) in a flat bottom 384-well tissue culture plate (Corning). Was plated in tissue culture medium at 500 cells / well. Cells were treated with a 4-fold serial dilution of the compound from 10,000 nM to 0.01 nΜ. Each concentration was tested in triplicate. At the time of treatment, the CellTiter-Glo® 2.0 luminescent cell viability assay was used according to the manufacturer's recommendations (Promega; # G9241) to evaluate plates of untreated cells. CellTiter-Glo® 2.0 reagent was added to the medium, incubated and assayed on the EnVision Multilabel Reader (PerkinElmer). The value represents the time point 0 (T0). The number of viable cells after 72 hours (T72) or 144 hours (T144) of compound treatment was also determined using the CellTiter-Glo® 2.0 luminescent cell viability assay. Emissions from time point 0 (T0), DMSO control growth (C), and test growth (T72 or T144) in the presence of the compound were used to calculate the growth rate at each compound concentration level. The growth inhibition rate is calculated as [(T144-T0) / (C-T0)] × 100 for the concentration of (for example) T144 ≧ T0 or [(T144-T0) / T0] × 100 for the concentration of T144 <T0. did. Prism 8 (Graphpad) and non-linear regression analysis and log (inhibitor) vs. A dose-response curve plot was created using a fitting with response-variable slope (4 parameters).

1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). FIG. 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. FIG. 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. FIG. 1C shows the viability dose response of HCC 1954 cells after incubation for 144 hours. FIG. 1D shows the viability dose response of NCI-N87 cells after incubation for 144 hours. Table 14 shows the GI50, LD50, and Rmin values for all the compounds tested.

4.1.2 Incel Splicing PD Assay SLC25A19 mature transcript splicing was also investigated in HCC1954 and NCI-N87 cells treated with an exemplary payload compound at increasing concentrations.

HCC 1954 or NCI-N87 cells (ATCC) were plated in RPMI + 10% FBS medium (ATCC) at 20 μL / well of 1000 cells / well. Cells were treated with the compound in a 4-fold diluted dose response. After 6 hours, cells are PBS washed, lysed in 30 μL CL buffer (IgePal CA-630, 5M NaCl, 1M Tris HCl in water 1M pH 7.4) containing 25 μL / mL RNAsin (Promega) and shaken. Motivated to incubate at room temperature for 20 minutes. Using the resulting mixture (1 μL), according to the manufacturer's recommendations, the following Taqman primers: SLC25A19 (Invitrogen, Hs00222265_m1); Taqman Fast Virus reverse transcription Master mix (A) with RPLPO (Invitrogen, Hs9999999902_m1). Splicing regulation was determined by PCR reaction.

2A and 2B show the results of splicing assays on HER2-amplified breast cancer cells (HCC1954) (FIG. 2A) and gastric cancer cells (NCI-N87) (FIG. 2B). Table 14 shows the IC50 values of all the compounds tested.

Claims (751)

Equation (I):
Ab- (L-H) _p (I)
[During the ceremony:
Ab is an antibody or antigen binding fragment that targets neoplastic cells;
H is a harboxydiene splicing regulator;
L is a linker that covalently binds Ab to H; and p is an integer of 1-15].
Antibodies-drug conjugates. The compound of formula (I) in which the harboxydiene splicing regulator is covalently linked to L via an arbitrary atom:

Or its pharmaceutically acceptable salt [in the formula:
Y is selected from O, S, NR ⁶ , and CR ⁶ R ⁷ ;
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁴ is hydrogen, C ₁ to C ₆ alkyl group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C. Selected from (= O)-(C ₃ to C ₈ heterocyclyl) groups and -C (= O) -NR ⁶ R ⁷ ;
R ⁵ is hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C (= O) -O- (C ₁ to C ₆ alkyl) group, -C (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , -OC (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , and -NR ⁶ -C (= O) -Selected from NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ and -C (= O) -OR ⁸ ; and R ⁸ is C ₁ Selected from ~ C ₆ alkyl groups, C ₃ ~ C ₈ carbocyclyl groups, and C ₃ ~ C ₈ heterocyclyl groups.
Here, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and —O— (C ₁ ). -C ₆ alkyl) group, -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ ). -C ₃ alkyl) and -NH-C (= O) -O- (may be independently substituted with 0 or 1 group selected from C ₁ to C ₃ alkyl)). The antibody-drug conjugate according to claim 1, wherein the atomic value is not exceeded, which is substituted with 0 to 3 groups and is covalently attached to L here. The compound of formula (Ia) in which the harboxydiene splicing regulator is covalently linked to L via an arbitrary atom:

Or its pharmaceutically acceptable salt [in the formula:
R ⁹ is selected from C ₃ to C ₈ heterocyclyl groups;
R ¹⁰ is selected from H and C ₁ to C ₆ alkyl groups.
Here, R ⁹ and R ¹⁰ are independently halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, -NH ₂ , -NH- (C ₁ to C ₃ alkyl), and -N- (C ₁ to C ₃ alkyl) substituted with 0 to 3 groups independently selected from ₂ , and where the valence of the atom covalently attached to L is not exceeded. ], The antibody-drug conjugate according to claim 1 or 2. The compound of formula (Ib) in which the harboxydiene splicing regulator is covalently linked to L via an arbitrary atom:

Or its pharmaceutically acceptable salt [in the formula:
R ¹¹ is

(In the formula, ^* represents the point where R ¹¹ binds to the rest of the compound);
R ¹² and R ¹³ are independently selected from H and methyl, respectively; and do not exceed the valence of said atom covalently attached to L, respectively], according to claims 1-3. The antibody-drug conjugate according to any one of the above. The harboxydiene splicing regulator is covalently linked to L via any atom.
The harboxydiene splicing regulator is selected from H1, H2, H3, and H12; and does not exceed the valence of the atom covalently attached to L, any of claims 1-4. The antibody-drug conjugate according to paragraph 1. L is covalently linked to the herboxydiene splicing regulator (“L—H”), and L—H has the following structure:

The antibody-drug conjugate according to claim 1 or 2, having a structure selected from the pharmaceutically acceptable salt thereof. Formula (II): The harboxydiene splicing regulator is covalently linked to L via any atom.

Or its pharmaceutically acceptable salt [in the formula:
X is hydroxyl or NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently hydrogen, -R ⁸ , -C (= O) -R ⁸ , -C (= O) -OR ⁸ ,-(C ₁ to C ₆ alkyl) -O. -C (= O) -R ⁸ and-(C ₁ to C ₆ alkyl) -NH-C (= O) -R ⁸ ; and R ⁸ is a C ₁ to C ₆ alkyl group, C ₃ Selected from the ~ _{C8 carbocyclyl} group and the C3 ~ _C8 heterocyclyl group.
Here, R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, -O- (C ₁ to C ₆ alkyl) groups, -CO ₂ H, and -C (, respectively. = O) -O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl Groups and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O). ) (C1 to C3 alkyl) and -NH _{- C (} = O) -O- ₍ C1 to C3 alkyl) may be independently substituted with 0 or 1 group selected from them). The splicing regulator according to claim 1, which is substituted with 0 to 3 groups independently selected from, and here does not exceed the valence of the atom covalently attached to L]. The antibody-drug conjugate described. Formula (IIa): The harboxydiene splicing regulator is covalently linked to L via any atom.

Or its pharmaceutically acceptable salt [in the formula:
Z is selected from NR ⁹ and O;
R ⁹ is selected from hydrogen and C ₁ to C ₆ alkyl groups;
R ¹⁰ and R ¹¹ are independently hydrogen, halogen, hydroxyl, C ₁ to C ₆ alkyl group, -O- (C ₁ to C ₆ alkyl) group, -CO ₂ H, -C (= O)-. O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl group. Selected from;
R ¹² is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups.
Here, R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, and C ₁ to C ₃ haloalkyl group, respectively. Substituted with 0 or 1 group to be;
t is an integer selected from 1, 2, 3, 4, 5, and 6; and here does not exceed the valence of the atom covalently attached to L] splicing regulator. The antibody-drug conjugate according to claim 1 or 7. Formula (IIb): The harboxydiene splicing regulator is covalently linked to L via any atom.

Or its pharmaceutically acceptable salt [in the formula:
R ¹³ is

( ^In the formula, ^* represents the point where R13 binds to the rest of the compound);
R ¹⁴ and R ¹⁵ are each independently selected from hydrogen and methyl; and here do not exceed the valence of said atom covalently attached to L], claim. The antibody-drug conjugate according to 1, 7, or 8. The herboxydiene splicing regulator is covalently linked to L via any atom;
The harboxydiene splicing regulator is selected from H4, H13, H14, H15, H16, H17, H18, H19, H20, H21, H23, and H24; and the atom covalently attached to L. The antibody-drug conjugate according to claim 1, 7, 8 or 9, which does not exceed the valence. L is covalently linked to the herboxydiene splicing regulator (“L—H”), and L—H has the following structure:

The antibody-drug conjugate according to claim 1 or 7, having a structure selected from the pharmaceutically acceptable salt thereof. Formula (III): The harboxydiene splicing regulator is covalently linked to L via any atom.

Or its pharmaceutically acceptable salt [in the formula:
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ , and -C (= O) -OR ⁸ ;
R ⁸ is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups; and R ⁹ is H,

Selected from;
Here, R ¹ , R ² , R ³ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and -O- (C ₁ to C ₆ alkyl) groups, respectively. , -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ ) ~ C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ ~ C ₈ carbocyclyl group, C ₁ ~ C ₆ alkyl hydroxy group, C ₁ ~ C ₆ alkyl alkoxy group, benzyl group, and C ₃ ~ C ₈ heterocyclyl group. (Each of these is halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ to C ₃ alkyl), And 0 to 3 independently selected from 0 or 1 group selected from -NH-C (= O) -O- (C ₁ to C ₃ alkyl)). Replaced by the group of
It does not exceed the valence of the atom covalently attached to L here; and in the formula, ^* represents the point where ^R9 binds to the rest of the compound] with the splicing regulator. The antibody-drug conjugate according to claim 1. The herboxydiene splicing regulator is covalently linked to L via any atom;
The harboxidien splicing regulator is selected from H5, H6, H7, H8, H9, H10, H11, H22, and H25; and can exceed the valence of the atom covalently attached to L. No, the antibody-drug conjugate according to claim 1 or 12. The linker is covalently linked to the harboxydiene splicing regulator (“L—H”), and L—H has the following structure:

The antibody-drug conjugate according to claim 1 or 12, having a structure selected from the pharmaceutically acceptable salt thereof. The herboxydiene splicing regulator is covalently linked to L via any atom;
The harboxydiene splicing regulators are H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21. , H22, H23, H24, and H25; and the antibody-drug conjugate according to claim 1, wherein the valence of the atom covalently attached to L is not exceeded. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H1. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H2. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H3. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H4. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H5. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H6. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H7. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H8. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H9. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H10. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H11. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H12. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H13. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H14. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H15. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H16. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H17. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H18. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H19. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H20. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H21. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H22. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H23. The antibody-drug conjugate according to claim 15, wherein the harboxydiene splicing regulator is H24. The antibody-drug conjugate according to any one of claims 1 to 39, wherein the linker is a cleavable linker. The antibody-drug conjugate according to claim 40, comprising a peptide moiety that can be cleaved by the linker. The antibody-drug conjugate according to claim 41, wherein the cleavable peptide moiety is enzymatically cleavable. The antibody-drug conjugate according to any one of claims 40 to 42, wherein the cleaving peptide moiety or linker comprises an amino acid unit. The antibody-drug conjugate according to claim 43, wherein the amino acid unit comprises valine-citrulline (Val-Cit). The antibody-drug conjugate according to claim 43, wherein the amino acid unit comprises valine-alanine (Val-Ala). The antibody-drug conjugate according to claim 43, wherein the amino acid unit comprises glutamine-valine-citrulline (Glu-Val-Cit). The antibody-drug conjugate according to claim 43, wherein the amino acid unit comprises alanine-alanine-asparagine (Ala-Ala-Asn). The antibody-drug conjugate according to claim 40, comprising a glucuronide moiety in which the linker can be cleaved. The antibody-drug conjugate according to claim 48, wherein the cleavable glucuronide moiety is enzymatically cleavable. The antibody-drug conjugate according to claim 48 or 49, wherein the cleavable glucuronide moiety is cleavable by glucuronidase. The antibody-drug conjugate according to any one of claims 48 to 50, wherein the cleavable glucuronide moiety is cleavable by β-glucuronidase. The antibody-drug conjugate according to any one of claims 1 to 51, wherein the linker comprises at least one spacer unit. 22. The antibody-drug conjugate according to claim 52, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. The antibody-drug conjugate according to claim 53, wherein the PEG moiety comprises-(PEG) _m -where m is an integer of 1-10. The antibody-drug conjugate according to claim 54, wherein m is 2. The antibody-drug conjugate according to claim 52, wherein the spacer unit or linker comprises an alkyl moiety. The antibody-drug conjugate according to claim 56, wherein the alkyl moiety comprises-(CH ₂ ) _n- , where n is an integer of 1-10. 58. The antibody-drug conjugate according to claim 57, wherein n is 2. 58. The antibody-drug conjugate according to claim 57, wherein n is 5. 58. The antibody-drug conjugate according to claim 57, wherein n is 6. The antibody-drug conjugate according to any one of claims 52 to 60, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). The antibody-drug conjugate according to claim 61, wherein the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigen binding fragment. The antibody-drug conjugate according to claim 61 or 62, wherein the Mal-spacer unit is conjugated to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 61 to 63, wherein the linker comprises the Mal-spacer unit and a cleavable peptide moiety. The antibody-drug conjugate according to claim 64, wherein the cleaving peptide moiety comprises an amino acid unit. The antibody-drug conjugate according to claim 64 or 65, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit. The antibody-drug conjugate according to claim 64 or 65, wherein the cleaving peptide moiety or amino acid unit comprises Val-Ala. The antibody-drug conjugate according to claim 64 or 65, wherein the cleaving peptide moiety or amino acid unit comprises Glu-Val-Cit. The antibody-drug conjugate according to claim 64 or 65, wherein the cleaving peptide moiety or amino acid unit comprises Ala-Ala-Asn. The antibody-drug conjugate according to any one of claims 61 to 69, wherein the Mal-spacer unit comprises an alkyl moiety. The antibody-drug conjugate according to any one of claims 61 to 69, wherein the Mal-spacer unit comprises a PEG moiety. The antibody-drug conjugate according to any one of claims 61 to 71, wherein the Mal-spacer unit comprises maleimide caproyl (MC). The antibody-drug conjugate according to any one of claims 61-72, wherein the Mal-spacer unit binds the antibody or antigen-binding fragment to the cleavable portion of the linker. The antibody-drug conjugate according to claim 73, wherein the cleavable portion of the linker comprises a cleavable peptide portion. The antibody-drug conjugate according to claim 74, wherein the cleaving peptide moiety comprises an amino acid unit. The antibody-drug conjugate according to claim 74 or 75, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. The antibody-drug conjugate according to any one of claims 73 to 76, wherein the linker comprises MC-Val-Cit. The antibody-drug conjugate according to any one of claims 73 to 76, wherein the linker comprises MC-Val-Ala. The antibody-drug conjugate according to any one of claims 73 to 76, wherein the linker comprises MC-Glu-Val-Cit. The antibody-drug conjugate according to any one of claims 73 to 76, wherein the linker comprises MC-Ala-Ala-Asn. The antibody-drug conjugate according to any one of claims 73 to 80, wherein the Mal-spacer unit comprises an alkyl moiety. The antibody-drug conjugate according to any one of claims 73 to 80, wherein the Mal-spacer unit comprises a PEG moiety. The antibody-drug conjugate according to any one of claims 73 to 82, wherein the Mal-spacer unit comprises maleimide caproyl (MC). 25. One of claims 52-83, wherein the cleavable portion of the linker is directly coupled to the splicing regulator, or a spacer unit binds the cleavable portion of the linker to the splicing regulator. Antibodies-drug conjugates. The antibody-drug conjugate according to claim 84, wherein cleavage of the conjugate releases the splicing regulator from the antibody or antigen binding fragment and linker. The antibody-drug conjugate according to claim 84 or 85, wherein the spacer unit that binds the cleavable portion of the linker to the splicing regulator is self-sacrificing. The antibody-drug conjugate according to any one of claims 84-86, wherein the spacer unit that binds the cleavable portion of the linker to the splicing regulator comprises p-aminobenzyloxycarbonyl (pABC). The antibody-drug conjugate according to claim 87, wherein the pABC binds the cleavable portion of the linker to the splicing regulator. The antibody-drug conjugate according to claim 87 or 88, wherein the cleavable portion of the linker comprises a cleavable peptide portion. The antibody-drug conjugate according to claim 89, wherein the cleaving peptide moiety comprises an amino acid unit. The antibody-drug conjugate according to claim 89 or 90, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. The antibody-drug conjugate according to any one of claims 87 to 91, wherein the linker comprises Val-Cit-pABC. The antibody-drug conjugate according to any one of claims 87 to 91, wherein the linker comprises Val-Ala-pABC. The antibody-drug conjugate according to any one of claims 87 to 91, wherein the linker comprises Glu-Val-Cit-pABC. The antibody-drug conjugate according to any one of claims 87-91, wherein the linker comprises Ala-Ala-Asn-pABC. The antibody-drug conjugate according to any one of claims 84-86, wherein the spacer unit for linking the cleavable portion of the linker to the splicing regulator comprises p-aminobenzyl (pAB). The antibody-drug conjugate according to claim 96, wherein the pAB binds the cleavable portion of the linker to the splicing regulator. The antibody-drug conjugate according to claim 96 or 97, wherein the cleavable portion of the linker comprises a cleavable peptide portion. The antibody-drug conjugate according to claim 98, wherein the cleaving peptide moiety comprises an amino acid unit. The antibody-drug conjugate according to claim 98 or 99, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. The antibody-drug conjugate according to any one of claims 96 to 100, wherein the linker comprises Val-Cit-pAB. The antibody-drug conjugate according to any one of claims 96 to 100, wherein the linker comprises Val-Ala-pAB. The antibody-drug conjugate according to any one of claims 96 to 100, wherein the linker comprises Glu-Val-Cit-pAB. The antibody-drug conjugate according to any one of claims 96 to 100, wherein the linker comprises Ala-Ala-Asn-pAB. The antibody-drug conjugate according to any one of claims 1 to 39, wherein the linker is a non-cleavable linker. The antibody-drug conjugate according to claim 105, wherein the linker comprises at least one spacer unit. The antibody-drug conjugate according to claim 105 or 106, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. The antibody-drug conjugate according to claim 107, wherein the PEG moiety comprises-(PEG) _m -where m is an integer of 1-10. The antibody-drug conjugate according to claim 108, wherein m is 2. The antibody-drug conjugate according to claim 105 or 106, wherein the spacer unit or linker comprises an alkyl moiety. The antibody-drug conjugate according to claim 110, wherein the alkyl moiety comprises-(CH ₂ ) _n- , where n is an integer of 1-10. The antibody-drug conjugate according to claim 111, wherein n is 2. The antibody-drug conjugate according to claim 111, wherein n is 5. The antibody-drug conjugate according to claim 111, wherein n is 6. The antibody-drug conjugate according to any one of claims 106 to 114, wherein the spacer unit is attached to the antibody or antigen binding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). The antibody-drug conjugate according to claim 115, wherein the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigen binding fragment. The antibody-drug conjugate according to claim 115 or 116, wherein the Mal-spacer unit is conjugated to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 115 to 117, wherein the Mal-spacer unit comprises an alkyl moiety. The antibody-drug conjugate according to any one of claims 115 to 117, wherein the Mal-spacer unit comprises a PEG moiety. The antibody-drug conjugate according to any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises maleimide caproyl (MC). The antibody-drug conjugate according to claim 120, wherein the linker or Mal-spacer unit comprises maleimide caproyl (MC) and at least one additional spacer unit. The antibody-drug conjugate according to any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises MC- (PEG) ₂ . The antibody-drug conjugate according to claim 122, wherein the linker or Mal-spacer unit comprises MC- (PEG) ₂ and at least one additional spacer unit. The antibody-drug conjugate according to any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises Mal-Hex. The antibody-drug conjugate according to claim 124, wherein the linker or Mal-spacer unit comprises Mal-Hex and at least one additional spacer unit. The antibody-drug conjugate according to any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises Mal-Et. The antibody-drug conjugate according to claim 126, wherein the linker or Mal-spacer unit comprises Mal-Et and at least one additional spacer unit. The antibody-drug conjugate according to any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises Mal-Et-O-Et. The antibody-drug conjugate according to claim 128, wherein the linker or Mal-spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit. The antibody-drug conjugate according to any one of claims 115 to 129, wherein the Mal-spacer unit binds the antibody or antigen-binding fragment to the splicing regulator. The antibody-drug conjugate according to any one of claims 115 to 130, wherein the linker comprises MC-Val-Cit-pABC. The antibody-drug conjugate according to any one of claims 115 to 130, wherein the linker comprises MC- (PEG) ₂ -CO. The antibody-drug conjugate according to any one of claims 1 to 132, wherein the antibody or antigen binding fragment targets neoplastic cells derived from a hematological malignancies or solid tumors. The antibody-drug conjugate according to any one of claims 1 to 133, wherein the antibody or antigen-binding fragment targets neoplastic cells derived from a hematological malignancies. The antibody-drug conjugate according to claim 133 or 134, wherein the hematological malignancy is selected from B cell malignancies, leukemias, lymphomas, and myelomas. The antibody-drug conjugate according to any one of claims 133 to 135, wherein the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. The antibody-drug conjugate according to any one of claims 1 to 133, wherein the antibody or antigen-binding fragment targets neoplastic cells derived from a solid tumor. The solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. The antibody-drug conjugate according to claim 133 or 137. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets HER2-expressing cells. The antibody-drug conjugate according to claim 139, wherein the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. Three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) in which the antibody or antigen binding fragment comprises the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3). And; claim comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3). 139 or 140 antibody-drug conjugates. 13. Antibodies-drug conjugates. The antibody-drug conjugate according to any one of claims 139 to 142, wherein the antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. The antibody-drug conjugate according to any one of claims 139 to 143, wherein the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets CD138-expressing cells. The antibody-drug conjugate according to claim 145, wherein the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. Three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) in which the antibody or antigen binding fragment comprises the amino acid sequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3). And; claim comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3). 145 or 146 antibody-drug conjugates. The invention according to any one of claims 145 to 147, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. Antibodies-drug conjugates. The antibody-drug conjugate according to any one of claims 145 to 148, wherein the antibody or antigen binding fragment comprises a human IgG2a heavy chain constant region. The antibody-drug conjugate according to any one of claims 145 to 149, wherein the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets EPHA2-expressing cells. 15. The antibody-drug conjugate of claim 151, wherein the antibody or antigen binding fragment is an anti-EPHA2 antibody or antigen binding fragment. Three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) in which the antibody or antigen binding fragment comprises the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3). And; claim comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3). 151 or 152 antibody-drug conjugates. The invention according to any one of claims 151 to 153, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. Antibodies-drug conjugates. The antibody-drug conjugate according to any one of claims 151 to 154, wherein the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. The antibody-drug conjugate according to any one of claims 151 to 155, wherein the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets MSLN-expressing cells. The antibody-drug conjugate according to claim 157, wherein the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets SOLH1-expressing cells. The antibody-drug conjugate according to claim 159, wherein the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets CDH6-expressing cells. The antibody-drug conjugate according to claim 161. The antibody or antigen-binding fragment is an anti-CDH6 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets CEACAM5-expressing cells. The antibody-drug conjugate according to claim 163, wherein the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets CFC1B expressing cells. The antibody-drug conjugate according to claim 165, wherein the antibody or antigen-binding fragment is an anti-CFC1B antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets ENPP3-expressing cells. The antibody-drug conjugate according to claim 167, wherein the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets FOLR1-expressing cells. The antibody-drug conjugate according to claim 169, wherein the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets HAVCR1-expressing cells. The antibody-drug conjugate according to claim 171. The antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets KIT-expressing cells. The antibody-drug conjugate according to claim 173, wherein the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets MET-expressing cells. The antibody-drug conjugate according to claim 175, wherein the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets MUC16 expressing cells. The antibody-drug conjugate according to claim 177, wherein the antibody or antigen binding fragment is an anti-MUC16 antibody or antigen binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets SLC39A6-expressing cells. The antibody-drug conjugate according to claim 179, wherein the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets SLC44A4 expressing cells. The antibody-drug conjugate according to claim 181. The antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 138, wherein the antibody or antigen binding fragment targets STEAP1-expressing cells. The antibody-drug conjugate according to claim 183, wherein the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment. The antibody-drug conjugate according to any one of claims 1 to 184, wherein p is 1 to 10. The antibody-drug conjugate according to any one of claims 1 to 185, wherein p is 2 to 8. The antibody-drug conjugate according to any one of claims 1 to 186, wherein p is 4 to 8. The antibody-drug conjugate according to any one of claims 1 to 187, wherein p is 4. The antibody-drug conjugate according to any one of claims 1 to 187, wherein p is 8. Compound of formula (I):

Or its pharmaceutically acceptable salt [in the formula:
Y is selected from O, S, NR ⁶ , and CR ⁶ R ⁷ ;
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁴ is hydrogen, C ₁ to C ₆ alkyl group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C. Selected from (= O)-(C ₃ to C ₈ heterocyclyl) groups and -C (= O) -NR ⁶ R ⁷ ;
R ⁵ is hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C (= O) -O- (C ₁ to C ₆ alkyl) group, -C (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , -OC (= O) -NR ⁶ R ⁷ , -NR ⁶ -C (= O) -R ⁸ , and -NR ⁶ -C (= O) -Selected from NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ and -C (= O) -OR ⁸ ; and R ⁸ is C ₁ Selected from ~ C ₆ alkyl groups, C ₃ ~ C ₈ carbocyclyl groups, and C ₃ ~ C ₈ heterocyclyl groups.
Here, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and —O— (C ₁ ). -C ₆ alkyl) group, -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ ). -C ₃ alkyl) and -NH-C (= O) -O- (may be independently substituted with 0 or 1 group selected from C ₁ to C ₃ alkyl)). It is replaced by 0 to 3 groups.] Compound of formula (Ia):

Or its pharmaceutically acceptable salt [in the formula:
R ⁹ is selected from the C ₃ to C ₈ heterocyclyl groups; and R ¹⁰ is selected from the H and C ₁ to C ₆ alkyl groups.
Here, R ⁹ and R ¹⁰ are independently halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, -NH ₂ , -NH- (C ₁ to C ₃ alkyl), and -N- (C ₁ to C ₃ Alkoxy) Substituted by 0 to 3 groups independently selected from ₂ ], according to claim 190. Compound of formula (Ib):

Or its pharmaceutically acceptable salt [in the formula:
R ¹¹ is

(In the formula, ^* represents the point where R ¹¹ binds to the rest of the compound); and R ¹² and R ¹³ are independently selected from H and methyl, respectively]. The compound according to claim 190 or 191. The compound according to claim 190, which is H1 or a pharmaceutically acceptable salt thereof. The compound according to claim 190, which is H2, or a pharmaceutically acceptable salt thereof. The compound according to claim 190, which is H3, or a pharmaceutically acceptable salt thereof. The compound according to claim 190, which is H12, or a pharmaceutically acceptable salt thereof. Equation (II):

Or its pharmaceutically acceptable salt [in the formula:
X is NR ⁶ R ⁷ ;
R ⁶ and R ⁷ are independently hydrogen, -R ⁸ , -C (= O) -R ⁸ , -C (= O) -OR ⁸ ,-(C ₁ to C ₆ alkyl) -O. -C (= O) -R ⁸ and-(C ₁ to C ₆ alkyl) -NH-C (= O) -R ⁸ ; and R ⁸ is a C ₁ to C ₆ alkyl group, C ₃ Selected from the ~ _{C8 carbocyclyl} group and the C3 ~ _C8 heterocyclyl group.
Here, R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, -O- (C ₁ to C ₆ alkyl) groups, -CO ₂ H, and -C (, respectively. = O) -O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl Groups and C ₃ to C ₈ heterocyclyl groups (each of which is a halogen, a hydroxyl, a C ₁ to C ₃ alkyl group, a C ₁ to C ₃ alkoxy group, a C ₁ to C ₃ haloalkyl group, -NH-C (= O). ) (C1 to C3 alkyl) and -NH _{- C (} = O) -O- ₍ C1 to C3 alkyl) may be independently substituted with 0 or 1 group selected from them). Substituted with 0 to 3 groups independently selected from]. Equation (IIa):

Or its pharmaceutically acceptable salt [in the formula:
Z is selected from NR ⁹ and O;
R ⁹ is selected from hydrogen and C ₁ to C ₆ alkyl groups;
R ¹⁰ and R ¹¹ are independently hydrogen, halogen, hydroxyl, C ₁ to C ₆ alkyl group, -O- (C ₁ to C ₆ alkyl) group, -CO ₂ H, -C (= O)-. O- (C ₁ to C ₆ alkyl) group, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ to C ₈ heterocyclyl) group, C ₃ to C ₈ carbocyclyl group, C ₁ to C ₆ alkyl hydroxy group, C ₁ to C ₆ alkyl alkoxy group, benzyl group, and C ₃ to C ₈ heterocyclyl group. Selected from;
R ¹² is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups.
Here, R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, and C ₁ to C ₃ haloalkyl group, respectively. The compound according to claim 197, wherein is substituted with 0 or 1 group to be; and t is an integer selected from 1, 2, 3, 4, 5, and 6. Equation (IIb):

Or its pharmaceutically acceptable salt [in the formula:
R ¹³ is

(In the formula, ^* represents the point where R ¹³ binds to the rest of the compound); and R ¹⁴ and R ¹⁵ are independently selected from hydrogen and methyl, respectively]. The compound according to claim 197 or 198. The compound according to claim 197, which is H4, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H13, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H14, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H15, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H16, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H17, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H18, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H19, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H20, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H21, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H23, or a pharmaceutically acceptable salt thereof. The compound according to claim 197, which is H24, or a pharmaceutically acceptable salt thereof. Compound of formula (III):

Or its pharmaceutically acceptable salt [in the formula:
R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl, -O- (C ₁ to C ₆ alkyl) groups, and -OC (= O)-(C ₁ to C ₆ alkyl) groups, respectively. , -C (= O) -O- (C ₁ to C ₆ alkyl) groups, and C ₁ to C ₆ alkyl groups;
R ⁶ and R ⁷ are independently selected from hydrogen, -R ⁸ , -C (= O) -R ⁸ , and -C (= O) -OR ⁸ ;
R ⁸ is selected from C ₁ to C ₆ alkyl groups, C ₃ to C ₈ carbocyclyl groups, and C ₃ to C ₈ heterocyclyl groups; and R ⁹ is H,

Selected from;
Here, R ¹ , R ² , R ³ , R ⁶ , R ⁷ , and R ⁸ are independently halogen, hydroxyl, C ₁ to C ₆ alkyl groups, and -O- (C ₁ to C ₆ alkyl) groups, respectively. , -CO ₂ H, -C (= O)-(C ₁ to C ₆ alkyl) group, -C (= O)-(C ₃ to C ₈ carbocyclyl) group, -C (= O)-(C ₃ ) ~ C ₈ heterocyclyl) group, -NR ⁶ R ⁷ , C ₃ ~ C ₈ carbocyclyl group, C ₁ ~ C ₆ alkyl hydroxy group, C ₁ ~ C ₆ alkyl alkoxy group, benzyl group, and C ₃ ~ C ₈ heterocyclyl group. (Each of these is halogen, hydroxyl, C ₁ to C ₃ alkyl group, C ₁ to C ₃ alkoxy group, C ₁ to C ₃ haloalkyl group, -NH-C (= O) (C ₁ to C ₃ alkyl), And 0 to 3 independently selected from 0 or 1 group selected from -NH-C (= O) -O- (C ₁ to C ₃ alkyl)). And in the formula, ^* represents the point where ^R9 binds to the rest of the compound]. The compound according to claim 212, which is H5, or a pharmaceutically acceptable salt thereof. The compound according to claim 212, which is H6, or a pharmaceutically acceptable salt thereof. The compound according to claim 212, which is H7, or a pharmaceutically acceptable salt thereof. The compound according to claim 212, which is H8, or a pharmaceutically acceptable salt thereof. The compound according to claim 212, which is H9, or a pharmaceutically acceptable salt thereof. The compound according to claim 212, which is H10, or a pharmaceutically acceptable salt thereof. The compound according to claim 212, which is H22, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising the compound according to any one of claims 190 to 219, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. A composition comprising a plurality of copies of the antibody-drug conjugate according to any one of claims 1 to 189, wherein the average p of the antibody-drug conjugate in the composition is about 3.5 to. The composition, which is about 5.5. 22. The composition of claim 221 which has an average p of the antibody-drug conjugate in the composition of about 4. A composition comprising a plurality of copies of the antibody-drug conjugate according to any one of claims 1 to 189, wherein the average p of the antibody-drug conjugate in the composition is about 7 to about 9. Is a composition. 223. The composition of claim 223, wherein the antibody-drug conjugate in the composition has an average p of about 8. The antibody-drug conjugate according to any one of claims 1 to 189, which is a method for treating a subject having or suspected to have a neoplastic disorder, and any of claims 190 to 219. A method comprising administering to the subject the compound according to claim 1, or the composition according to any one of claims 220 to 224. 225. The method of claim 225, wherein the neoplastic disorder is a hematological malignancies or solid tumors. 225. The method of claim 225 or 226, wherein the neoplastic disorder is a hematological malignancies. 227. The method of claim 227, wherein the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. 225. The method of claim 225 or 226, wherein the neoplastic disorder is a solid tumor. The solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. , The method of claim 229. The method according to any one of claims 225 to 230, wherein the subject has one or more neoplastic cells expressing the target antigen. 231. The method of claim 231. The target antigen is HER2. 231 or 232 of the method of claim 231 or 232, wherein the one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer expressing HER2. 233. The method of claim 233, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial cancer. The subject according to any one of claims 232 to 234, wherein the subject is (a) refractory or refractory to treatment with an anti-HER2 antibody when administered alone and / or (b) a splicing regulator when administered alone. The method described. The method according to any one of claims 232 to 235, wherein the subject is intolerant, refractory, or refractory to treatment with a splicing regulator when administered alone. 231. The method of claim 231. The target antigen is CD138. 231 or 237. The method of claim 231 or 237, wherein the one or more neoplastic cells are derived from multiple myeloma expressing CD138. 237 or 238. The method of claim 237 or 238, wherein the subject is (a) refractory or refractory to treatment with an anti-CD138 antibody when administered alone and / or (b) a splicing regulator when administered alone. The method according to any one of claims 237 to 239, wherein the subject is intolerant, refractory, or refractory to treatment with a splicing regulator when administered alone. 231. The method of claim 231. The target antigen is EPHA2. 231 or 241 of the method of claim 231 or 241 wherein the one or more neoplasmic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2. 241 or 242. The method of claim 241 or 242, wherein the subject is refractory or refractory to treatment with (a) an anti-EPHA2 antibody when administered alone and / or (b) a splicing regulator when administered alone. The method according to any one of claims 241 to 243, wherein the subject is intolerant, refractory, or refractory to treatment with a splicing regulator when administered alone. The antibody-drug conjugate according to any one of claims 1 to 189, which is a method for reducing or inhibiting tumor growth in a subject having or suspected of having a neoplastic disorder. A method comprising administering to the subject the compound according to any one of items 190 to 219 or the composition according to any one of claims 220 to 224. 245. The method of claim 245, wherein the tumor comprises one or more neoplasmic cells expressing the target antigen. 246. The method of claim 246, wherein the target antigen is HER2. The method of claim 246 or 247, wherein the one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer expressing HER2. 248. The method of claim 248, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial cancer. 13. the method of. 246. The method of claim 246, wherein the target antigen is CD138. 246 or 251 of the method of claim 246, wherein the one or more neoplastic cells are derived from multiple myeloma expressing CD138. 25. The method of claim 251 or 252, wherein the tumor is (a) resistant or refractory to treatment with an anti-CD138 antibody when administered alone and / or (b) a splicing regulator when administered alone. 246. The method of claim 246, wherein the target antigen is EPHA2. 246 or 254 according to claim 246 or 254, wherein the one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2. 25. The method of claim 254 or 255, wherein the tumor is (a) resistant or refractory to treatment with an anti-EPHA2 antibody when administered alone and / or (b) a splicing regulator when administered alone. The antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, or a claim to a subject having or suspected of having a neobiological disorder. A method for determining whether or not the treatment with the composition according to any one of items 220 to 224 is effective, wherein a biological sample from the subject is provided, and any one of claims 1 to 189. The antibody-drug conjugate according to claim, the compound according to any one of claims 190 to 219, or the composition according to any one of claims 220 to 224 is brought into contact with the biological sample. How to include. 257. The method of claim 257, wherein the biological sample is a tumor sample. 258. The method of claim 258, wherein the tumor sample is a tumor biopsy or blood sample. 259. The method of claim 259, wherein the blood sample is selected from blood, a blood fraction, or cells obtained from the blood or blood fraction. The method according to any one of claims 257 to 260, wherein the subject has one or more neoplastic cells expressing the target antigen. 261. The method of claim 261, wherein the target antigen is HER2. 261. The method of claim 261 or 262, wherein the one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary tract cancer expressing HER2. 263. The method of claim 263, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial cancer. 261. The method of claim 261, wherein the target antigen is CD138. 261 or 265. The method of claim 261 or 265, wherein the one or more neoplastic cells are derived from multiple myeloma expressing CD138. 261. The method of claim 261, wherein the target antigen is EPHA2. 261 or 267. The method of claim 261 or 267, wherein the one or more neoplasmic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2. The antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, the compound according to claim 220 to, for use in the treatment of neoplastic disorders. The composition according to any one of 224. The antibody-drug conjugate, compound, or composition for use according to claim 269, wherein the neoplastic disorder is characterized by having one or more neoplastic cells expressing the target antigen. The antibody-drug conjugate, compound, or composition for use according to claim 270, wherein the target antigen is HER2. The antibody-drug conjugate for use according to claim 270 or 271, wherein the neoplastic disorder is HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer. , Compounds, or compositions. 272. The antibody-drug conjugate, compound, or composition of claim 272, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial cancer. The antibody-drug conjugate, compound, or composition for use according to claim 270, wherein the target antigen is CD138. The antibody-drug conjugate, compound, or composition for use according to claim 270 or 274, wherein the neoplastic disorder is multiple myeloma expressing CD138. The antibody-drug conjugate, compound, or composition for use according to claim 270, wherein the target antigen is EPHA2. The antibody-drug conjugate for use according to claim 270 or 276, wherein the neoplastic disorder is breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that express EPHA2. , Compounds, or compositions. The antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, or any of claims 220 to 224 in the treatment of neoplastic disorders. Use of the composition according to claim 1. The use according to claim 278, wherein the neoplastic disorder is characterized by having one or more neoplastic cells expressing the target antigen. The use according to claim 279, wherein the target antigen is HER2. The use according to claim 279 or 280, wherein the neoplastic disorder is HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer. The use according to claim 281, wherein the lung cancer is a lung adenocarcinoma and / or the uterine cancer is a serous endometrial cancer. The use according to claim 279, wherein the target antigen is CD138. The use according to claim 279 or 283, wherein the neoplastic disorder is multiple myeloma expressing CD138. The use according to claim 279, wherein the target antigen is EPHA2. The use according to claim 279 or 285, wherein the neoplastic disorder is breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that express EPHA2. The antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, or the compound according to any one of claims 190 to 219, in a method for producing a pharmaceutical product for treating a neoplastic disorder. Use of the composition according to any one of 220 to 224. The use according to claim 287, wherein the neoplastic disorder is characterized by having one or more neoplastic cells expressing the target antigen. The use according to claim 288, wherein the target antigen is HER2. The use according to claim 288 or 289, wherein the neoplastic disorder is HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer. The use according to claim 290, wherein the lung cancer is a lung adenocarcinoma and / or the uterine cancer is a serous endometrial cancer. The use according to claim 288, wherein the target antigen is CD138. The use according to claim 288 or 292, wherein the neoplastic disorder is multiple myeloma expressing CD138. The use according to claim 288, wherein the target antigen is EPHA2. The use according to claim 288 or 294, wherein the neoplastic disorder is breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that express EPHA2. The preparation of an antibody-drug conjugate according to any one of claims 1 to 189, which comprises reacting an antibody or antigen-binding fragment with a linker coupled to a splicing regulator under conditions that allow conjugation. Method. The antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, which is a method for inducing at least one neoantigen. Alternatively, a method comprising contacting the composition according to any one of claims 220 to 224 with neoplastic cells thereby inducing the production of at least one neoantigen. 297. The method of claim 297, wherein the neoplastic cells are present in an in vitro cell culture. The method of claim 297 or 298, wherein the neoplastic cells are obtained from the subject. 297. The method of claim 297, wherein the neoplastic cells are present in the subject. The method according to any one of claims 297 to 300, wherein the neoplasmic cells are derived from a hematological malignant tumor or a solid tumor. The method of claim 301, wherein the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. 30. The method of claim 301 or 302, wherein the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. The solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. , The method of claim 301. The antibody according to any one of claims 1 to 189, which is a method for inducing at least one neoantigen and / or T cell response in a subject having or suspected of having a neoplastic disorder. A method comprising administering to said subject a drug conjugate, the compound according to any one of claims 190 to 219, or the composition according to any one of claims 220 to 224. The antibody-drug conjugate according to any one of claims 1 to 189, which is a method for treating a subject having or suspected to have a neoplastic disorder, and any of claims 190 to 219. The administration of the compound according to one or the composition according to any one of claims 220 to 224 is included, and administration of the compound, antibody-drug conjugate, or composition is at least one. A method of inducing a neoantigen and / or T cell response. The dose of the compound, antibody-drug conjugate, or composition administered is at least one neoantigen and / or when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. The method of claim 305 or 306, wherein the induction of T cell response is reduced. The dosage of the compound, antibody-drug conjugate, or composition is 10%, 15%, 20%, 25 when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. The method of any one of claims 305-307, wherein the method is reduced by%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%. At least 10%, 15%, 20%, 25%, when the compound, antibody-drug conjugate, or composition is compared to the standard dosing regimen of the compound, antibody-drug conjugate, or composition. The method of any one of claims 305-308, which is administered at a frequency of 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently. 13. Method. The method of any one of claims 305-310, further comprising administering at least one additional therapy. The dose of the compound, antibody-drug conjugate, composition, and / or the at least one additional therapy is that of the compound, antibody-drug conjugate, composition, and / or the at least one additional therapy. The method of claim 311, wherein the induction of at least one neoantigen and / or T cell response is reduced when compared to a standard dosage. The dose of the compound, antibody-drug conjugate, composition, and / or the at least one additional therapy is that of the splicing regulator, antibody-drug conjugate, composition, and / or the at least one additional therapy. 30%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% reduction when compared to standard dosages, claim 311. Or the method according to 312. The compound, antibody-drug conjugate, composition, and / or the at least one additional therapy is the standard dosing regimen of the compound, antibody-drug conjugate, composition, and / or the at least one additional therapy. 311 to claim 311 to which are administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently when compared to. The method according to any one of 313. 31-11 to claim that the dosage and / or dosage of the compound, antibody-drug conjugate, composition, and / or at least one additional therapy results in reduced systemic toxicity and / or improved tolerability. The method according to any one of 314. The method of any one of claims 311-315, wherein administration of the compound, antibody-drug conjugate, or composition is initiated prior to administration of the at least one additional therapy. The method of any one of claims 311-315, wherein administration of the compound, antibody-drug conjugate, or composition is initiated after administration of the at least one additional therapy. The method of any one of claims 311-315, wherein administration of the compound, antibody-drug conjugate, or composition is initiated simultaneously with administration of the at least one additional therapy. The method of any one of claims 305-318, wherein administration of the compound, antibody-drug conjugate, or composition is repeated at least once after the initial administration. 319. The method of claim 319, wherein the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the amount used for initial administration. Claim that the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. 319 or 320. The amount used for repeated administration of the compound, antibody-drug conjugate, or composition is 10%, 15% when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% reduction, according to any one of claims 319-321. The method according to any one of claims 311 to 322, wherein the administration of the at least one additional therapy is repeated at least once after the initial administration. 323. The method of claim 323, wherein the amount used for repeated doses of the at least one additional therapy is reduced when compared to the amount used for the initial dose. 323 or 324. The method of claim 323 or 324, wherein the amount used for repeated administration of the at least one additional therapy is reduced when compared to a standard dosage of the at least one additional therapy. The amount used for repeated doses of the at least one additional therapy is 10%, 15%, 20%, 25%, 30%, 35% when compared to the standard dosage of the at least one additional therapy. , 40%, 45%, 50%, 75%, or 90%, according to any one of claims 323 to 325. The method of any one of claims 319-326, wherein repeated administration of the compound, antibody-drug conjugate, or composition is simultaneous with repeated administration of the at least one additional therapy. One of claims 319-326, wherein repeated administration of the compound, antibody-drug conjugate, or composition is sequential or staggered with repeated administration of the at least one additional therapy. The method described. The method of any one of claims 311-328, wherein the at least one additional therapy comprises administering a checkpoint inhibitor. 329. The method of claim 329, wherein the subject is intolerant, intolerant, or refractory to the checkpoint inhibitor when administered alone. 30. The method of claim 329 or 330, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR. The method of any one of claims 329-331, wherein the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR. The method according to any one of claims 329 to 332, wherein the checkpoint inhibitor comprises a cytotoxic T lymphocyte-related antigen 4 pathway (CTLA4) inhibitor. 333. The method of claim 333, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody. 334. The method of claim 334, wherein the anti-CTLA4 antibody is ipilimumab. The method of any one of claims 329-332, wherein the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor. 336. The method of claim 336, wherein the PD1 inhibitor is an anti-PD1 antibody. 337. The method of claim 337, wherein the anti-PD1 antibody is nivolumab. 336. The method of claim 336, wherein the PD1 inhibitor is an anti-PDL1 antibody. 339. The method of claim 339, wherein the anti-PDL1 antibody is atezolizumab. The method according to any one of claims 329 to 332, wherein the checkpoint inhibitor comprises a CTLA4 inhibitor and a PD1 inhibitor. 341. The method of claim 341, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody. 342. The method of claim 342, wherein the anti-CTLA4 antibody is ipilimumab. The method of claim 341, wherein the PD1 inhibitor is an anti-PD1 antibody. 344. The method of claim 344, wherein the anti-PD1 antibody is nivolumab. The method of claim 341, wherein the PD1 inhibitor is an anti-PDL1 antibody. 346. The method of claim 346, wherein the anti-PDL1 antibody is atezolizumab. The method of any one of claims 311-328, wherein the at least one additional therapy comprises administering a neoantigen vaccine. 348. The method of claim 348, wherein the compound, antibody-drug conjugate, or composition is administered prior to administration of the neoantigen vaccine. 348. The method of claim 348, wherein the compound, antibody-drug conjugate, or composition is administered after administration of the neoantigen vaccine. 348. The method of claim 348, wherein the compound, antibody-drug conjugate, or composition is administered at the same time as administration of the neoantigen vaccine. The method of any one of claims 348-351, wherein administration of the compound, antibody-drug conjugate, or composition is repeated at least once after the initial administration. 352. The method of claim 352, wherein the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the amount used for initial administration. The method according to any one of claims 348 to 353, wherein the neoantigen vaccine comprises at least one neoantigen peptide. 354. The method of claim 354, wherein the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. The method of claim 354 or 355, wherein the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length. The method according to any one of claims 354 to 356, wherein the at least one neoantigen peptide comprises one or more neoantigen sequences. The method of claim 357, wherein the neoantigen sequence is a neoantigen sequence specific to the subject. The method of claim 357, wherein the neoantigen sequence is a universal neoantigen sequence. 358. The method of claim 358, wherein the neo-antigen sequence is a neo-antigen vaccine personalized for the subject. The neoantigen sequence has been identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition. The method according to 360. The method of claim 360 or 361, wherein the neoantigen sequence has the ability to bind at least one HLA allele expressed in the subject. 359. The method of claim 359, wherein the neoantigen sequence is a universal neoantigen vaccine. The neoantigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% of the subjects in the subject population suffering from the neoplasmic disorder. 363. The method of claim 363, which has the ability to bind at least one HLA allele that is expressed in at least 45%. 363. 364. 354-365. The method described in any one of the items. 348. The method of claim 348, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier. The method of claim 367, wherein the at least one neoantigen peptide is linked to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. 368. The method according to any one of claims 367 to 369, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are covalently linked via a linker. The method according to any one of claims 367 to 369, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein. 348. The method of claim 348, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent. 348. The method of claim 348, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable adjuvant. 366. The method of claim 366, wherein the neoplastic cells are present in an in vitro cell culture. 364. The method of claim 366 or 374, wherein the neoplastic cells are obtained from the subject. 366. The method of claim 366, wherein the neoplastic cells are present in the subject. The method according to any one of claims 348 to 353, wherein the neoantigen vaccine comprises at least one neoantigen mRNA. 377. The method of claim 377, wherein the at least one neoantigen mRNA encodes one or more neoantigen sequences. The method of claim 378, wherein the neoantigen sequence is a neoantigen sequence specific to the subject. The method of claim 378, wherein the neoantigen sequence is a universal neoantigen sequence. 379. The method of claim 379, wherein the neo-antigen sequence is a neo-antigen vaccine personalized for the subject. The neoantigen sequence has been identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition. 381. 381. The method of claim 381 or 382, wherein the neoantigen sequence has the ability to bind at least one HLA allele expressed in the subject. The method of claim 380, wherein the neoantigen sequence is a universal neoantigen vaccine. The neoantigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% of the subjects in the subject population suffering from the neoplasmic disorder. Or the method of claim 384, which has the ability to bind at least one HLA allele expressed in at least 45%. The neoantigen sequence is capable of inducing a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of the subject population suffering from the neoplasmic disorder, claim 384 or. 385. Any of claims 378-386, wherein the at least one neoantigen mRNA encodes a neoantigen sequence induced by contacting an effective amount of the compound, antibody-drug conjugate, or composition with a neoplastic cell. The method described in item 1. 377. The method of claim 377, wherein the neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable carrier. 388. The method of claim 388, wherein the at least one neoantigen mRNA is ligated to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. 389. 377. The method of claim 377, wherein the neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable diluent. 377. The method of claim 377, wherein the neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable adjuvant. The method according to any one of claims 377 to 392, wherein the neoantigen mRNA is encapsulated in an encapsulant. 393. The method of claim 393, wherein the encapsulant is a liposome. 393. The method of claim 393, wherein the encapsulant is nanoparticles. 387. The method of claim 387, wherein the neoplastic cells are present in an in vitro cell culture. 387. The method of claim 387 or 396, wherein the neoplastic cells are obtained from the subject. 387. The method of claim 387, wherein the neoplastic cells are present in the subject. The method of any one of claims 311-328, wherein the at least one additional therapy comprises administering a cytokine or cytokine analog. 399. The method of claim 399, wherein the subject is intolerant, intolerant, or refractory to the cytokine or cytokine analog when administered alone. The method of claim 399 or 400, wherein the cytokine or cytokine analog comprises a T cell enhancer. The method of any one of claims 399-401, wherein the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα. The method of any one of claims 311-328, wherein the at least one additional therapy comprises administering modified tumor-targeted T cells. The invention of any one of claims 305-403, further comprising detecting one or more neoantigens and / or T cell responses in the subject after administration of the compound, antibody-drug conjugate, or composition. the method of. 404. The method of claim 404, further comprising continuing administration of the compound, antibody-drug conjugate, or composition if one or more neoantigens and / or T cell responses are detected. If one or more neoantigens and / or T cell responses are detected, further comprising continuing administration of the compound, antibody-drug conjugate, or composition at a lower frequency and / or reduced dosage. , The method of claim 404 or 405. One of claims 404-406, wherein detection of one or more neoantigens and / or T cell responses in the subject dictates the effectiveness of treatment with the compound, antibody-drug conjugate, or composition. The method described in the section. The method of any one of claims 305-407, wherein the subject has a non-synonymous mutation load of about 150 or less mutations. The method of any one of claims 305-408, wherein the subject has a non-synonymous mutation load of about 100 or less mutations. The method of any one of claims 305-409, wherein the subject has a non-synonymous mutation load of about 50 or less mutations. The method according to any one of claims 305 to 410, wherein the neoplastic disorder is a hematological malignant tumor or a solid tumor. 411. The method of claim 411, wherein the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. The method of claim 411 or 412, wherein the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. The solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. , The method of claim 411. A method of treating a subject who has or is suspected of having a neoplastic disorder.
(A) An effective amount of the antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, or any one of claims 220 to 224. Administration of the composition according to the section to said subject, wherein administration of the compound, antibody-drug conjugate, or composition induces at least one neoantigen and / or T cell response;
(B) Detecting one or more neoantigens and / or T cell responses in said subject after administration of the compound, antibody-drug conjugate, or composition; and (c) one or more neoantigens and /. Alternatively, a method comprising continuing administration of the compound, antibody-drug conjugate, or composition if a T cell response is detected. 415. The method of claim 415, wherein detection of one or more neoantigens and / or T cell responses in the subject dictates the effectiveness of treatment with the compound, antibody-drug conjugate, or composition. A method for treating a subject having or suspected of having a neoplastic disorder, wherein an effective amount of the antibody-drug conjugate according to any one of claims 1 to 189, any of claims 190 to 219. A method comprising administering to said subject the compound according to one or the composition according to any one of claims 220 to 224; and at least one additional therapy. 417. The method of claim 417, wherein the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies. 417 or 418. The method of claim 417 or 418, wherein administration of the compound, antibody-drug conjugate, or composition induces at least one neoantigen and / or T cell response. A dose of said compound, antibody-drug conjugate, or composition and / or said at least one additional therapy is standard for said compound, antibody-drug conjugate, or composition and / or said at least one additional therapy. 417-419, which are reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to different dosages. The method described in any one of the above. The compound, antibody-drug conjugate, or composition and / or the at least one additional therapy is the standard dosing regimen of the compound, antibody-drug conjugate, or composition and / or the at least one additional therapy. 417-claim, which are administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently when compared to. The method according to any one of 420. 417 to claim that the dosage and / or dosage of the compound, antibody-drug conjugate, or composition and / or at least one of the additional therapies results in reduced systemic toxicity and / or improved tolerability. The method according to any one of 421. The method of any one of claims 417-422, wherein administration of the compound, antibody-drug conjugate, or composition is initiated prior to administration of the at least one additional therapy. The method of any one of claims 417-422, wherein administration of the compound, antibody-drug conjugate, or composition is initiated after administration of the at least one additional therapy. The method of any one of claims 417-422, wherein administration of the compound, antibody-drug conjugate, or composition is initiated simultaneously with administration of the at least one additional therapy. The method of any one of claims 417-425, wherein administration of the compound, antibody-drug conjugate, or composition is repeated at least once after the initial administration. 426. The method of claim 426, wherein the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the amount used for initial administration. Claim that the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. 426 or 427. The amount used for repeated administration of the compound, antibody-drug conjugate, or composition is 10%, 15% when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% reduction, according to any one of claims 426-428. The method of any one of claims 417-429, wherein administration of the at least one additional therapy is repeated at least once after the initial administration. 430. The method of claim 430, wherein the amount used for repeated doses of the at least one additional therapy is reduced when compared to the amount used for the initial dose. The method of claim 430 or 431, wherein the amount used for repeated administration of the at least one additional therapy is reduced when compared to the standard dosage of the at least one additional therapy. The amount used for repeated doses of the at least one additional therapy is 10%, 15%, 20%, 25%, 30%, 35% when compared to the standard dosage of the at least one additional therapy. , 40%, 45%, 50%, 75%, or 90%, according to any one of claims 430 to 432. The method of any one of claims 426-433, wherein repeated administration of the compound, antibody-drug conjugate, or composition is simultaneous with repeated administration of the at least one additional therapy. 13. The method described. The method of any one of claims 417-435, wherein the at least one additional therapy comprises administering a checkpoint inhibitor. 436. The method of claim 436, wherein the subject is intolerant, intolerant, or refractory to the checkpoint inhibitor when administered alone. 436 or 437, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR. The method of any one of claims 436-438, wherein the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR. The method according to any one of claims 436 to 439, wherein the checkpoint inhibitor comprises a cytotoxic T lymphocyte-related antigen 4 pathway (CTLA4) inhibitor. 440. The method of claim 440, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody. 441. The method of claim 441, wherein the anti-CTLA4 antibody is ipilimumab. The method of any one of claims 436-439, wherein the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor. 443. The method of claim 443, wherein the PD1 inhibitor is an anti-PD1 antibody. 444. The method of claim 444, wherein the anti-PD1 antibody is nivolumab. 443. The method of claim 443, wherein the PD1 inhibitor is an anti-PDL1 antibody. 446. The method of claim 446, wherein the anti-PDL1 antibody is atezolizumab. The method according to any one of claims 436 to 439, wherein the checkpoint inhibitor comprises a CTLA4 inhibitor and a PD1 inhibitor. The method of claim 448, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody. 449. The method of claim 449, wherein the anti-CTLA4 antibody is ipilimumab. The method of claim 448, wherein the PD1 inhibitor is an anti-PD1 antibody. 451. The method of claim 451 wherein the anti-PD1 antibody is nivolumab. The method of claim 448, wherein the PD1 inhibitor is an anti-PDL1 antibody. 453. The method of claim 453, wherein the anti-PDL1 antibody is atezolizumab. The method of any one of claims 417-435, wherein the at least one additional therapy comprises administering a neoantigen vaccine. 455. The method of claim 455, wherein the compound, antibody-drug conjugate, or composition is administered prior to administration of the neoantigen vaccine. 455. The method of claim 455, wherein the compound, antibody-drug conjugate, or composition is administered after administration of the neoantigen vaccine. 455. The method of claim 455, wherein the compound, antibody-drug conjugate, or composition is administered at the same time as administration of the neoantigen vaccine. The method of any one of claims 455-458, wherein administration of the compound, antibody-drug conjugate, or composition is repeated at least once after the initial administration. 459. The method of claim 459, wherein the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the amount used for initial administration. The method according to any one of claims 455 to 460, wherein the neoantigen vaccine comprises at least one neoantigen peptide. 461. The method of claim 461, wherein the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. 461. The method of claim 461, wherein the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length. The method according to any one of claims 461 to 463, wherein the at least one neoantigen peptide comprises one or more neoantigen sequences. 464. The method of claim 464, wherein the neoantigen sequence is a neoantigen sequence specific to the subject. 464. The method of claim 464, wherein the neoantigen sequence is a universal neoantigen sequence. 465. The method of claim 465, wherein the neo-antigen sequence is a neo-antigen vaccine personalized for the subject. The neoantigen sequence has been identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition. 467. The method of claim 467 or 468, wherein the neoantigen sequence has the ability to bind at least one HLA allele expressed in the subject. 466. The method of claim 466, wherein the neoantigen sequence is a universal neoantigen vaccine. The neoantigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% of the subjects in the subject population suffering from the neoplasmic disorder. Or the method of claim 470, which has the ability to bind at least one HLA allele expressed in at least 45%. The neoantigen sequence is capable of inducing a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of the subject population suffering from the neoplasmic disorder, claim 470 or. 471. Any of claims 464-472, wherein the at least one neoantigen peptide comprises a neoantigen sequence derived by contacting an effective amount of the compound, antibody-drug conjugate, or composition with a neoplastic cell. The method described in paragraph 1. 455. The method of claim 455, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier. 474. The method of claim 474, wherein the at least one neoantigen peptide is linked to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. 475. The method according to any one of claims 474 to 476, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are covalently linked via a linker. The method according to any one of claims 474 to 476, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein. The method of claim 455, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent. 455. The method of claim 455, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable adjuvant. 473. The method of claim 473, wherein the neoplastic cells are present in an in vitro cell culture. 473 or 481. The method of claim 473 or 481, wherein the neoplastic cells are obtained from the subject. 473. The method of claim 473, wherein the neoplastic cells are present in the subject. The method of any one of claims 455-460, wherein the neo-antigen vaccine comprises at least one neo-antigen mRNA. 484. The method of claim 484, wherein the at least one neoantigen mRNA encodes one or more neoantigen sequences. The method of claim 485, wherein the neoantigen sequence is a neoantigen sequence specific to the subject. The method of claim 485, wherein the neoantigen sequence is a universal neoantigen sequence. 486. The method of claim 486, wherein the neo-antigen sequence is a neo-antigen vaccine personalized for the subject. The neoantigen sequence has been identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition. 488. The method of claim 488 or 489, wherein the neoantigen sequence has the ability to bind at least one HLA allele expressed in the subject. 487. The method of claim 487, wherein the neoantigen sequence is a universal neoantigen vaccine. The neoantigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% of the subjects in the subject population suffering from the neoplasmic disorder. Or the method of claim 491, which has the ability to bind at least one HLA allele expressed in at least 45%. The neoantigen sequence is capable of inducing a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of the subject population suffering from the neoplasmic disorder, claim 491 or. 492. Any of claims 484-493, wherein the at least one neoantigen mRNA encodes a neoantigen sequence induced by contacting an effective amount of the compound, antibody-drug conjugate, or composition with a neoplastic cell. The method described in item 1. 484. The method of claim 484, wherein the neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable carrier. 495. The method of claim 495, wherein the at least one neoantigen mRNA is ligated to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines. The method according to 496. 484. The method of claim 484, wherein the neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable diluent. 484. The method of claim 484, wherein the neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable adjuvant. The method according to any one of claims 484 to 499, wherein the neoantigen mRNA is encapsulated in an encapsulant. The method of claim 500, wherein the encapsulant is a liposome. The method of claim 500, wherein the encapsulant is nanoparticles. 494. The method of claim 494, wherein the neoplastic cells are present in an in vitro cell culture. The method of claim 494 or 503, wherein the neoplastic cells are obtained from the subject. 494. The method of claim 494, wherein the neoplastic cells are present in the subject. The method of any one of claims 417-435, wherein the at least one additional therapy comprises administering a cytokine or cytokine analog. 506. The method of claim 506, wherein the subject is intolerant, intolerant, or refractory to the cytokine or cytokine analog when administered alone. The method of claim 506 or 507, wherein the cytokine or cytokine analog comprises a T cell enhancer. The method according to any one of claims 506 to 508, wherein the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα. The method of any one of claims 417-435, wherein the at least one additional therapy comprises administering modified tumor-targeted T cells. The method of any one of claims 417-510, wherein the subject has a non-synonymous mutation load of about 150 or less mutations. The method of any one of claims 417-511, wherein the subject has a non-synonymous mutation load of about 100 or less mutations. The method of any one of claims 417-512, wherein the subject has a non-synonymous mutation load of about 50 or less mutations. The method according to any one of claims 417 to 513, wherein the neoplastic disorder is a hematological malignant tumor or a solid tumor. 514. The method of claim 514, wherein the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. The method of claim 514 or 515, wherein the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. The solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. , The method of claim 514. A method for identifying at least one neoantigen,
(A) An effective amount of the antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, or any one of claims 220 to 224. Contacting the composition described in the section with neoplastic cells;
(B) Detecting at least one selectively spliced mRNA transcript after contacting the compound, antibody-drug conjugate, or composition with the neoplastic cells;
(C) Predicting translation of the at least one selectively spliced mRNA transcript into at least one peptide; and (d) comparing the at least one peptide with a reference proteome, said. A method comprising identifying at least one neoantigen if at least one peptide does not match any peptide of the reference proteome. 518. The method of claim 518, wherein detecting at least one selectively spliced mRNA transcript comprises RNAseq. Predicting the translation of the at least one selectively spliced mRNA transcript comprises quantifying the change in percent splice-in (dPSI) value for the at least one transcript, claim 518 or 519. The method described in. The method of any one of claims 518-520, wherein predicting translation of the at least one selectively spliced mRNA transcript comprises RiboSeq and / or ribosome profiling. The method of any one of claims 518-521, further comprising assessing the at least one peptide for predicted major histocompatibility complex (MHC) binding. 522. The method of claim 522, wherein the predicted MHC binding is determined by measuring the uncorrected affinity predicted binding strength of the at least one peptide. 523. The method of claim 523, wherein the uncorrected affinity predicted binding strength of about 500 nM or higher indicates MHC binding. Any of claims 522-524, wherein the predicted MHC binding is determined by identifying a distribution of predicted binding strengths for a series of random peptides; and comparing the predicted binding strengths of the at least one peptide to the distribution. The method described in paragraph 1. 525. The method of claim 525, which exhibits an MHC bond with a weak predicted bond strength in the top 2% of the distribution. 525. The method of claim 525, which exhibits an MHC bond with a strong predicted bond strength in the top 0.5% of the distribution. The method of any one of claims 518-527, wherein the neoplastic cells are present in an in vitro cell culture. 528. The method of claim 528, wherein the neoplastic cells are obtained from the subject. The method according to any one of claims 518 to 527, wherein the neoplastic cells are present in the subject. The method of any one of claims 518-530, further comprising contacting with one or more additional neoplastic cells to identify at least one universal neoantigen. A method for identifying at least one neoantigen,
(A) An effective amount of the antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, or any one of claims 220 to 224. Contacting the composition described in the section with neoplastic cells;
(B) After contacting the compound, antibody-drug conjugate, or composition with the neogenic cell, detection of at least one peptide containing a potential neoantigen sequence; and (c) at least one of the above. A method of comparing a peptide to a reference proteome, comprising identifying at least one neoantigen if the at least one peptide does not match any peptide of the reference proteome. 532. The method of claim 532, further comprising assessing the at least one peptide for predicted major histocompatibility complex (MHC) binding. 533. The method of claim 533, wherein the predicted MHC binding is determined by measuring the uncorrected affinity predicted binding strength of the at least one peptide. 534. The method of claim 534, wherein the uncorrected affinity predicted binding strength of about 500 nM or higher indicates MHC binding. Any of claims 533-535, wherein the predicted MHC binding is determined by identifying a distribution of predicted binding strengths for a series of random peptides; and comparing the predicted binding strengths of the at least one peptide to the distribution. The method described in paragraph 1. 536. The method of claim 536, which exhibits an MHC bond with a weak predicted bond strength in the top 2% of the distribution. 536. The method of claim 536, which exhibits an MHC bond with a strong predicted bond strength in the top 0.5% of the distribution. The method of any one of claims 532-538, wherein the neoplastic cells are present in an in vitro cell culture. The method of claim 539, wherein the neoplastic cells are obtained from the subject. The method according to any one of claims 532 to 538, wherein the neoplastic cells are present in the subject. The method of any one of claims 532-541, further comprising contacting with one or more additional neoplastic cells to identify at least one universal neoantigen. It is a method of making a neo-antigen vaccine.
(A) Identifying at least one neoantigen using the method according to any one of claims 518-542; and (b) pharmaceutically acceptable carrier, dilution of the at least one neoantigen. A method comprising formulating with an agent or adjuvant. 543. The method of claim 543, wherein the at least one neoantigen is linked to the pharmaceutically acceptable carrier. 544. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. The method described. A method of treating a subject who has or is suspected of having a neoplastic disorder.
(A) An effective amount of the antibody-drug conjugate according to any one of claims 1 to 189, the compound according to any one of claims 190 to 219, or any one of claims 220 to 224. Administering the composition according to the section to the subject;
(B) Detection of one or more neoantigens in the subject after administration of the compound, antibody-drug conjugate, or composition;
(C) Comparing the one or more neoantigens with a panel of universal neoantigens; and (d) administering to the subject a universal neoantigen vaccine comprising at least one universal neoantigen present in the subject. How to include. 546. The method of claim 546, wherein the universal neoantigen vaccine is administered alone or in combination with at least one additional therapy. 547. The method of claim 547, wherein the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies. 547 or 548. The method of claim 547 or 548, wherein the at least one additional therapy comprises repeated administration of the compound, antibody-drug conjugate, or composition. 549. The method of claim 549, wherein repeated administration of the compound, antibody-drug conjugate, or composition is initiated prior to administration of the universal neoantigen vaccine. 549. The method of claim 549, wherein the repetition of the compound, antibody-drug conjugate, or composition is initiated after administration of the universal neoantigen vaccine. 549. The method of claim 549, wherein repeated administration of the compound, antibody-drug conjugate, or composition is initiated simultaneously with administration of the universal neoantigen vaccine. The one of claims 549-552, wherein the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the amount used for initial administration. Method. Claimed that the amount used for repeated administration of the compound, antibody-drug conjugate, or composition is reduced when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. The method according to any one of 549 to 553. The amount used for repeated administration of the compound, antibody-drug conjugate, or composition is 10%, 15% when compared to the standard dosage of the compound, antibody-drug conjugate, or composition. , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% reduction, according to any one of claims 549-554. The method of any one of claims 547-555, wherein the at least one additional therapy comprises administering a checkpoint inhibitor. 556. The method of claim 556, wherein administration of the checkpoint inhibitor is initiated prior to administration of the universal neoantigen vaccine and / or prior to repeated administration of the compound, antibody-drug conjugate, or composition. 556. The method of claim 556, wherein administration of the checkpoint inhibitor is initiated after administration of the universal neoantigen vaccine and / or after repetition of the compound, antibody-drug conjugate, or composition. 556. The method of claim 556, wherein administration of the checkpoint inhibitor is initiated simultaneously with administration of the universal neoantigen vaccine and / or repeated administration of the compound, antibody-drug conjugate, or composition. The method according to any one of claims 556 to 559, wherein the administration of the checkpoint inhibitor is repeated at least once after the initial administration. 560. The method of claim 560, wherein the amount used for repeated doses of the checkpoint inhibitor is reduced when compared to the amount used for the initial dose. 560 or 561. The method of claim 560 or 561, wherein the amount used for repeated administration of the checkpoint inhibitor is reduced when compared to a standard dosage of the checkpoint inhibitor. The amount used for repeated doses of the checkpoint inhibitor is 10%, 15%, 20%, 25%, 30%, 35%, 40 when compared to the standard dosage of the checkpoint inhibitor. The method according to any one of claims 560 to 562, wherein the method is reduced by%, 45%, 50%, 75%, or 90%. The method according to any one of claims 556 to 563, wherein the subject is intolerant, refractory, or refractory to the checkpoint inhibitor when administered alone. The method of any one of claims 556-564, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR. The method of any one of claims 556-565, wherein the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR. The method according to any one of claims 556 to 566, wherein the checkpoint inhibitor comprises a cytotoxic T lymphocyte-related antigen 4 pathway (CTLA4) inhibitor. 567. The method of claim 567, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody. 568. The method of claim 568, wherein the anti-CTLA4 antibody is ipilimumab. The method of any one of claims 556-566, wherein the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor. The method of claim 570, wherein the PD1 inhibitor is an anti-PD1 antibody. 571. The method of claim 571, wherein the anti-PD1 antibody is nivolumab. The method of claim 570, wherein the PD1 inhibitor is an anti-PDL1 antibody. 573. The method of claim 573, wherein the anti-PDL1 antibody is atezolizumab. The method according to any one of claims 556 to 566, wherein the checkpoint inhibitor comprises a CTLA4 inhibitor and a PD1 inhibitor. 575. The method of claim 575, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody. 576. The method of claim 576, wherein the anti-CTLA4 antibody is ipilimumab. The method of claim 575, wherein the PD1 inhibitor is an anti-PD1 antibody. 578. The method of claim 578, wherein the anti-PD1 antibody is nivolumab. The method of claim 575, wherein the PD1 inhibitor is an anti-PDL1 antibody. 580. The method of claim 580, wherein the anti-PDL1 antibody is atezolizumab. The method of any one of claims 546-581, wherein the universal neoantigen vaccine comprises at least one neoantigen peptide. 582. The method of claim 582, wherein the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. 582. The method of claim 582 or 583, wherein the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length. The method according to any one of claims 582 to 584, wherein the at least one neoantigen peptide comprises one or more universal neoantigen sequences. The universal neo-antigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% of the subjects in the subject population suffering from the neoplasmic disorder. , Or the method of claim 585, which has the ability to bind at least one HLA allele expressed in at least 45%. Claim 585, wherein the universal neoantigen sequence has the ability to elicit a T cell response to tumors present in at least 1%, at least 5%, or at least 10% of the subject population suffering from the neoplasmic disorder. Or the method according to 586. 582. The method of claim 582, wherein the universal neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier. 588. The method of claim 588, wherein the at least one neoantigen peptide is linked to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines. 589. The method according to any one of claims 588 to 590, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are covalently linked via a linker. The method according to any one of claims 588 to 590, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein. 582. The method of claim 582, wherein the universal neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent. 582. The method of claim 582, wherein the universal neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable adjuvant. The method of any one of claims 546-581, wherein the universal neoantigen vaccine comprises at least one neoantigen mRNA. 595. The method of claim 595, wherein the at least one neoantigen mRNA encodes one or more universal neoantigen sequences. The universal neo-antigen sequence is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% of the subjects in the subject population suffering from the neoplasmic disorder. , Or the method of claim 596, which has the ability to bind at least one HLA allele expressed in at least 45%. Claim 596, wherein the universal neoantigen sequence has the ability to elicit a T cell response to tumors present in at least 1%, at least 5%, or at least 10% of the subject population suffering from the neoplasmic disorder. Or the method according to 597. 595. The method of claim 595, wherein the universal neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable carrier. 599. The method of claim 599, wherein the at least one neoantigen mRNA is ligated to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines. The method according to 600. 595. The method of claim 595, wherein the universal neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable diluent. 595. The method of claim 595, wherein the universal neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable adjuvant. The method according to any one of claims 595 to 603, wherein the neoantigen mRNA is encapsulated in an encapsulant. 604. The method of claim 604, wherein the encapsulant is a liposome. 604. The method of claim 604, wherein the encapsulant is nanoparticles. The method of any one of claims 546-606, wherein the subject has a non-synonymous mutation load of about 150 or less mutations. The method of any one of claims 546-607, wherein the subject has a non-synonymous mutation load of about 100 or less mutations. The method of any one of claims 546-608, wherein the subject has a non-synonymous mutation load of about 50 or less mutations. The method according to any one of claims 546 to 609, wherein the neoplastic disorder is a hematological malignant tumor or a solid tumor. 610. The method of claim 610, wherein the hematological malignancies are selected from B cell malignancies, leukemias, lymphomas, and myelomas. The method of claim 610 or 611, wherein the hematological malignancies are selected from acute myeloid leukemia and multiple myeloma. The solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. , The method of claim 610. A neoantigen vaccine comprising at least one neoantigen peptide, wherein the at least one neoantigen peptide is an effective amount of the antibody-drug conjugate according to any one of claims 1 to 189, claim 190 to. A neoantigen vaccine comprising a neoantigen sequence derived by contacting a neoantigen cell with the compound according to any one of 219 or the composition according to any one of claims 220 to 224. The neoantigen vaccine according to claim 614, wherein the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. The neoantigen vaccine according to claim 614 or 615, wherein the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length. The neoantigen vaccine according to any one of claims 614 to 616, further comprising a pharmaceutically acceptable carrier. 617. The neoantigen vaccine according to claim 617, wherein the at least one neoantigen peptide is linked to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. 618. Neoantigen vaccine. The neoantigen vaccine according to any one of claims 617 to 619, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are covalently linked via a linker. The neoantigen vaccine according to any one of claims 617 to 619, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein. The neoantigen vaccine according to any one of claims 614 to 616, further comprising a pharmaceutically acceptable diluent. The neoantigen vaccine according to any one of claims 614 to 616, further comprising a pharmaceutically acceptable adjuvant. The neoantigen vaccine according to any one of claims 614 to 623, wherein the neoplastic cells are present in an in vitro cell culture. The neoantigen vaccine according to any one of claims 614 to 624, wherein the neoplastic cells are obtained from the subject. The neoantigen vaccine according to any one of claims 614 to 623, wherein the neoplastic cells are present in the subject. A neoantigen vaccine comprising at least one neoantigen mRNA, wherein the at least one neoantigen mRNA is an effective amount of the antibody-drug conjugate according to any one of claims 1 to 189, claim 190 to. A neoantigen vaccine encoding a neoantigen sequence induced by contacting a neoantigen cell with the compound according to any one of 219 or the composition according to any one of claims 220 to 224. The neoantigen vaccine of claim 627, further comprising a pharmaceutically acceptable carrier. The neoantigen vaccine according to claim 628, wherein the at least one neoantigen mRNA is ligated to the pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocianine, immunoglobulins, tyroglobulin, ovalbumin, toxoid or attenuated toxoid derivatives, cytokines, and chemokines. 629. Neoantigen vaccine. The neoantigen vaccine of claim 627, further comprising a pharmaceutically acceptable diluent. The neoantigen vaccine of claim 627, further comprising a pharmaceutically acceptable adjuvant. The neoantigen vaccine according to any one of claims 627 to 632, wherein the neoantigen mRNA is encapsulated in an encapsulant. The neoantigen vaccine according to claim 633, wherein the encapsulant is a liposome. The neoantigen vaccine according to claim 633, wherein the encapsulant is nanoparticles. The neoantigen vaccine according to any one of claims 627 to 635, wherein the neoplastic cells are present in an in vitro cell culture. The neoantigen vaccine according to any one of claims 627 to 636, wherein the neoplastic cells are obtained from the subject. The neoantigen vaccine according to any one of claims 627 to 635, wherein the neoplastic cells are present in the subject. The compound according to any one of claims 190 to 219 or the pharmaceutical composition according to claim 220, which is a method for treating a subject having or suspected of having a neoplastic disorder. Methods involving administration to a subject. The compound according to any one of claims 190 to 219 or the compound according to claim 220, which is a method for reducing or inhibiting the growth of a tumor in a subject having or suspected of having a neoplastic disorder. A method comprising administering the pharmaceutical composition of the above to the subject.

And its pharmaceutically acceptable salts [in the formula, L is a linker covalently attached to the antibody].

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof.

The compound according to claim 641, which is a pharmaceutically acceptable salt thereof. The compound according to any one of claims 641 to 678, wherein the linker is a cleavable linker. The compound according to claim 679, which comprises a peptide moiety that can be cleaved by the linker. The compound according to claim 680, wherein the cleavable peptide moiety is enzymatically cleavable. The compound according to any one of claims 679 to 681, wherein the cleaving peptide moiety or linker comprises an amino acid unit. The compound according to claim 682, wherein the amino acid unit comprises valine-citrulline (Val-Cit). The compound according to claim 682, wherein the amino acid unit comprises valine-alanine (Val-Ala). The compound according to claim 682, wherein the amino acid unit comprises glutamine-valine-citrulline (Glu-Val-Cit). The compound according to claim 682, wherein the amino acid unit comprises alanine-alanine-asparagine (Ala-Ala-Asn). The compound according to claim 679, which comprises a glucuronide moiety in which the linker can be cleaved. The compound according to claim 687, wherein the cleaveable glucuronide moiety is enzymatically cleaveable. The compound according to claim 687 or 688, wherein the cleaveable glucuronide moiety is cleaveable by glucuronidase. The compound according to any one of claims 687 to 689, wherein the cleaveable glucuronide moiety can be cleaved by β-glucuronidase. The compound according to any one of claims 641 to 690, wherein the linker comprises at least one spacer unit. The compound according to claim 691, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. 692. The compound of claim 692, wherein the PEG moiety comprises − (PEG) _m −, where m is an integer of 1-10. The compound according to claim 693, wherein m is 2. The compound of claim 693, wherein the spacer unit or linker comprises an alkyl moiety. The compound according to claim 695, wherein the alkyl moiety comprises-(CH ₂ ) _n- , where n is an integer of 1-10. The compound according to claim 696, wherein n is 2. The compound according to claim 696, wherein n is 5. The compound according to claim 696, wherein n is 6. The compound according to any one of claims 691 to 699, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). The compound according to claim 700, wherein the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigen binding fragment. The compound according to claim 700 or 701, wherein the Mal-spacer unit is conjugated to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment. The compound according to any one of claims 700 to 702, wherein the linker comprises the Mal-spacer unit and a cleavable peptide moiety. The compound according to claim 703, wherein the cleaving peptide moiety comprises an amino acid unit. The compound according to claim 703 or 704, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit. The compound according to claim 703 or 704, wherein the cleaving peptide moiety or amino acid unit comprises Val-Ala. The compound according to claim 703 or 704, wherein the cleaving peptide moiety or amino acid unit comprises Glu-Val-Cit. The compound according to claim 703 or 704, wherein the cleaving peptide moiety or amino acid unit comprises Ala-Ala-Asn. The compound according to any one of claims 700 to 708, wherein the Mal-spacer unit comprises an alkyl moiety. The compound according to any one of claims 700 to 708, wherein the Mal-spacer unit comprises a PEG moiety. The compound according to any one of claims 700 to 710, wherein the Mal-spacer unit comprises maleimide caproyl (MC). The compound according to any one of claims 700 to 711, wherein the Mal-spacer unit binds the antibody or antigen-binding fragment to the cleavable portion of the linker. 712. The compound according to claim 712, wherein the cleavable moiety of the linker comprises a cleavable peptide moiety. The compound according to claim 713, wherein the cleaving peptide moiety comprises an amino acid unit. The compound according to claim 713 or 714, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. The compound according to any one of claims 712 to 715, wherein the linker comprises MC-Val-Cit. The compound according to any one of claims 712 to 715, wherein the linker comprises MC-Val-Ala. The compound according to any one of claims 712 to 715, wherein the linker comprises MC-Glu-Val-Cit. The compound according to any one of claims 712 to 715, wherein the linker comprises MC-Ala-Ala-Asn. The compound according to any one of claims 712 to 719, wherein the Mal-spacer unit comprises an alkyl moiety. The compound according to any one of claims 712 to 719, wherein the Mal-spacer unit comprises a PEG moiety. The compound according to any one of claims 712 to 721, wherein the Mal-spacer unit comprises maleimide caproyl (MC). 601-722. Compound. The compound according to claim 723, wherein cleavage of the conjugate releases the splicing regulator from the antibody or antigen binding fragment and linker. The compound according to claim 723 or 724, wherein the spacer unit that binds the cleavable portion of the linker to the splicing regulator is self-sacrificing. The compound according to any one of claims 723 to 725, wherein the spacer unit for linking the cleavable portion of the linker to the splicing regulator comprises p-aminobenzyloxycarbonyl (pABC). 726. The compound of claim 726, wherein the pABC binds the cleavable portion of the linker to the splicing regulator. The compound according to claim 726 or 727, wherein the cleaveable portion of the linker comprises a cleaveable peptide portion. The compound according to claim 728, wherein the cleaving peptide moiety comprises an amino acid unit. The compound according to claim 728 or 729, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. The compound according to any one of claims 726 to 730, wherein the linker comprises Val-Cit-pABC. The compound according to any one of claims 726 to 730, wherein the linker comprises Val-Ala-pABC. The compound according to any one of claims 726 to 730, wherein the linker comprises Glu-Val-Cit-pABC. The compound according to any one of claims 726 to 730, wherein the linker comprises Ala-Ala-Asn-pABC. The compound according to any one of claims 723 to 725, wherein the spacer unit for linking the cleavable portion of the linker to the splicing regulator comprises p-aminobenzyl (pAB). 735. The compound of claim 735, wherein the pAB binds the cleavable portion of the linker to the splicing regulator. The compound according to claim 735 or 736, wherein the cleaveable portion of the linker comprises a cleaveable peptide portion. The compound according to claim 737, wherein the cleaving peptide moiety comprises an amino acid unit. The compound according to claim 737 or 738, wherein the cleaving peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. The compound according to any one of claims 735 to 739, wherein the linker comprises Val-Cit-pAB. The compound according to any one of claims 735 to 739, wherein the linker comprises Val-Ala-pAB. The compound according to any one of claims 735 to 739, wherein the linker comprises Glu-Val-Cit-pAB. The compound according to any one of claims 735 to 739, wherein the linker comprises Ala-Ala-Asn-pAB. The compound according to any one of claims 641 to 678, wherein the linker is a non-cleavable linker. The compound according to claim 744, wherein the linker comprises at least one spacer unit. The compound according to claim 744 or 745, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. 746. The compound of claim 746, wherein the PEG moiety comprises − (PEG) _m −, where m is an integer of 1-10. The compound according to claim 747, wherein m is 2. The compound according to claim 744 or 745, wherein the spacer unit or linker comprises an alkyl moiety. The compound according to claim 749, wherein the alkyl moiety comprises-(CH ₂ ) _n- , where n is an integer of 1-10. A method for producing an antibody-drug conjugate, which comprises reacting the compound according to any one of claims 641 to 750 with an antibody or an antigen-binding fragment under conditions that allow conjugation.

Images